Sortilin 1 is a novel inducer of vascular calcification

ABSTRACT

The invention relates to methods for decreasing, inhibiting, preventing, or reducing calcification by inhibiting sortilin 1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 14/439,951 filed on Apr. 30, 2015, which is a 371 National PhaseEntry of International Patent Application No. PCT/US2013/067969 filed onNov. 1, 2013 which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/721,727 filed Nov. 2, 2012, the contentsof which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name 14439,951.txt, creation date of Aug. 13, 2017 and a size of2,182 bytes. The sequence listing submitted via EFS-Web is part of thespecification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to methods and compositions fordecreasing, inhibiting, treating, or preventing cardiovascularcalcification, e.g., arterial and valvular microcalcification.

BACKGROUND

Vascular and valvular calcification, a prominent feature of chronicinflammatory disorders such as chronic renal disease, type II diabetesand dyslipidemia, associates with significant morbidity and mortality.Clinical, histological, and animal studies suggest that processes invascular calcification are similar to those of bone remodeling (Hyder JA et al, American Journal of Epidemiology, 2009; Lieberman M et al,Arteriosclerosis, Thrombosis, and Vascular Biology, 2008; Bucay N et al,Genes & Development, 1998; Khosla S et al, Nature Medicine, 2011).Vascular calcification is an active, cell-regulated process in whichvascular smooth muscle cells (SMCs) can lose the expression of theirmarker genes, acquire osteogenic markers, and deposit a mineralizedbone-like matrix (Bostrom K I et al, Circulation Research, 2011). SMCsmay play an important role in this process via transition toward anosteoblast-like state or releasing calcified matrix vesicles andmicroparticles.

Various therapeutic agents have been investigated to targetcardiovascular calcification; these include statins (Aikawa E et al,Circulation, 2007; Monzack et al, ATVB, 2009; Osman L et al,Circulation, 2006; Rajamannan N M et al, Circulation, 2005; Wu Y W etal, Eur J Nucl Med Mol Imaging, 2012), bisphosphonate (Hartle J E et al,Am J Kidney Dis, 2012), phosphate binder (Di Iorio B et al, Clin J AmSoc Nephrol, 2012) and mineralocorticoid receptor antagonists (Gkizas Set al, Cardiovasc Pharma, 2010; Jaffe I Z et al, ATVB, 2007). However,beneficial effects of these drugs remain uncertain in the clinicalsetting (Gilmanov D, Inter. Cardiovasc Thor Surg, 2010). Thus, despiteglobal clinical burden of cardiovascular calcification, no medicaltherapies are available.

Calcification in coronary arteries promotes heart attacks, whichrepresent major health problems and economic burden in the UnitedStates. Calcification in carotid arteries associate with risk for strokeand dementia. Calcification in aortic valves causes aortic stenosis andheart failure. Especially, patients with mineral imbalance andcalcium/phosphate disorders, including chronic renal disease,hemodyalysis and type II diabetes suffer from accelerated vascular andvalvular calcification. For instance, arterio-venous shunts/grafts forhemodialysis in patients with chronic renal disease, vein grafts forperipheral arterial disease in diabetic patients, and saphenous veinbypass grafts for occluded coronary arteries in patients with metabolicdisorders are often occluded within a year (vein graft failure). In thefuture, tissue engineered vascular and valvular implants in patients atmetabolic risk may often fail. If the vein graft for peripheral arterialdisease fails, the only options is an expensive redo surgery or stentimplantation, or devastating amputation of the lower extremity. However,despite its high clinical and economic impact, no medical therapies areavailable to prevent or treat calcification.

SUMMARY

In part, this invention is based on inventors' discovery that sortilin 1unexpectedly is an inducer of vascular calcification via aphosphate-dependent mechanism. The inventors have discovered inter aliathat sortilin 1 is a novel regulator of hyperphosphatemia-triggeredvascular calcification. As demonstrated herein, sortilin 1 promotes anosteogenic phenotype of SMC and may induce the release and calcificationpotential of matrix vesicles in a calciying environment. Without wishingto be bound by a theory, blocking sortilin 1 function by preventing itsassociation with binding partners, inhibits calcification. The datapresented herein shows that exogenous sortilin 1 increases calcificationand serum sortilin 1 levels are elevated in atherosclerotic mice.Without limitations, neutralizing antibodies can be used to interferethe sortilin 1-mediated pro-calcific pathway. Small molecules and RNAican also be used.

Accordingly, in one aspect provided herein is method of decreasing,inhibiting, preventing, or reducing calcification of SMCs. Generally,the method comprises contacting SMCs with a compound that can inhibitthe activity or amount of sortilin 1 in SMCs. Alternatively, the methodcontacting SMCs with a compound that can inhibit the expression of anucleic acid encoding sortilin 1 in the SMC.

The method described herein can be used for inhibiting, decreasing,preventing, or treating vascular calcification in a subject byadministering a compound to the subject in need thereof, wherein thecompound decreases, inhibits, prevents or reduces: (i) activity oramount of sortilin 1 in SMC; or (ii) expression of a nucleic acidencoding sortilin 1 in SMC; or (iii) phosphorylation of sortilin 1. Insome embodiments, the compounds inhibits or reduces phosphorylation ofserine 819 or/and 825 of sortilin 1. In some embodiments, saidadministering can be orally, intravenously, subcutaneously or locally.

In some embodiments, cardiovascular calcification is valvular orarterial microcalcification/gross calcification.

In some embodiments, the subject has chronic renal disease, severe renalfailure treated with hemodialysis, hemodialysis AV grafts/shunts, veingrafts, vascular anastomsis, Paget's disease, diabetes, dyslipidemia,osteoarthritis, rheumatoid athritis or osteoporosis.

In some embodiments, the subject has a transcatheter valve implant.

In some embodiments, the subject has chronic coronary atherosclerosis oraortic stenosis.

In some embodiments, the nucleic acid encoding sortilin 1 is SORT 1 geneor SORT 1 mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show that Sortilin 1 is induced in human calcified tissueand during osteogenic transition of human vascular SMCs. FIG. 1A,Sortilin 1 (left) is expressed in calcified regions of humanatherosclerotic carotid arteries. Purple staining by hematoxylinindicated calcified area. SMCs are stained with alpha smooth muscle cellactin (αSMA, middle) and macrophages with CD68 (right). One out of 20patient samples is shown. Bar; 100 μm. FIG. 1B, Immunofluorescencemicroscopy shows the co-expression of sortilin 1 (left) and RUNX2(middle) in human calcified lesion. Nuclei stained with DAPI (right).Bar; 50 μm. FIGS. 1C-1E, SMCs were cultured up to 21 days in controlmedium (CM) or osteogenic medium (OM). FIG. 1C, Sortilin 1 mRNAexpression, relative to day 1, n=4 independent cell donor. *p<0.05. FIG.1D, Sortilin 1 protein expression. Top: representative Western blot,bottom: statistical analysis, relative to day 1, n=4 independent celldonor. *p<0.05. FIG. 1E, Immunofluorescence microscopy shows theexpression of sortilin 1 and osteopontin (OPN) in calcified SMCs (day21). Bar; 100 μm. Error bars indicate SD.

FIGS. 2A-2G show that modification of sortilin 1 alters alkalinephosphatase activity and matrix mineralization in calcifying SMCs. SMCswere cultured for 14 (alkaline phosphatase activity) or 21 (matrixmineralization) days in control medium (CM), or osteogenic medium (OM).Sortilin 1 was either silenced by siRNA (A, B, D; siSORT or scramblecontrol, Scr) or cells were stimulated with recombinant sortilin 1 (B,E; rSORT1, 200 ng/ml) twice per week during the entire cell cultureperiod. FIGS. 2A and 2B, Tissue non-specific alkaline phosphataseactivity (TNAP). FIG. 2C, Representative image of bright field SMCsshowing the nodule formation. FIGS. 2D and 2E, Matrix mineralization.Top: Representative images of alizarin Red S-stained mineralized matrix.n=4 independent cell donor. FIGS. 2F and 2G, SMCs were cultured inosteogenic medium. Sortilin 1 was overexpressed by adenovirus (AdSORT1).LacZ served as control (AdLacZ). FIG. 2F, TNAP and FIG. 2G, Matrixmineralization. n=3 independent cell donor. Error bars indicate SD.

FIGS. 3A-3E show that Sortilin 1 regulates PHEX expression. SMCs werecultured for 21 days in control medium (CM), or osteogenic medium (OM).Sortilin 1 was either silenced by siRNA (siSORT1 or scramble control,Scr), overexpressed by adenovirus (AdSORT1 or AdLacZ) or cells werestimulated with recombinant sortilin 1 (rSORT1, 200 ng/ml) twice perweek during the entire cell culture period. FIG. 3A, Cell viability atday 3 in osteogenic medium. n=3 independent cell donor. FIG. 3B, PHEXsnRNA expression in calcified SMCs determined using PCR array. n=4independent cell donor. FIGS. 3C and 3D, PHEX expression. Top:representative Western blot, b-actin served as loading control. Bottom:Quantification of mRNA expression. n=4 independent cell donor; *p<0.05.FIG. 3E, Correlation of PHEX and sortilin 1 mRNA expression. r=−0.865;R²=0.748; p=0.0001. Error bars indicate SD.

FIGS. 4A-4E show that miR125b regulates sortilin 1 by direct binding.FIG. 4A, the binding site for miR-125b within the SORT1 3′UTR is highlyconserved between different species. Analysis was done usingwww.targetscan.com at Apr. 30, 2012. Sequence shown is AAAUGCUCAGGGUCCCC(SEQ ID NO: 1). FIGS. 4B-4D, SMCs were cultured in control medium (CM),or osteogenic medium (OM) and transfected with miR-125b inhibitor(Anti-125b), and the corresponding control (Scr) for 72 hours. FIG. 4B,miR-125b expression. FIG. 4C, Sortilin 1 expression. Top: representativeWestern blot, b-actin served as loading control. Bottom: mRNAexpression. n=4 independent cell donor; *p<0.05. FIG. 4D, Correlation ofmiR-125b expression and sortilin 1 mRNA expression. r=−0.825; R²=0.681;p=0.043. FIG. 4E, SORT1 3′UTR reporter activity is repressed by miR125bmimic (Pre-miR-125b) and induced by miR125b inhibitor (Anti-miR125b).Data are given as fold increase from the corresponding control. n=3independent cell donor. *p<0.05. Error bars indicate SD.

FIGS. 5A-5G show that Sortilin 1 is increased in chronic renal disease,induced by 5/6 nephrectomy in Apoe−/− mice. FIG. 5A, Immunofluorescencestaining of sortilin 1 in wild type (WT) and Apoe−/− mice without andwith (CDR) 5/6 nephrectomy. Sortilin 1; red. DAPI; blue,autofluorescence; green. One of four animals per group is shown. Bar; 50μm. FIG. 5B, Immunohistochemical staining of sortilin 1 in aorta of WT,Apoe−/− mice without and with chronic renal disease (CRD) 5/6nephrectomy. One of four animals per group is shown. Magnification: 40×.FIGS. 5C and 5D, Quantification of sortilin 1 positive cells inatherosclerotic lesion. Separate analysis of vessel media (C) and intima(D). n=4. *p<0.05. FIG. 5E Sortilin 1 serum levels. n=3-5. FIG. 5F,FGF23 serum levels. n=3-5. FIG. 5G, Correlation of serum sortilin 1 andserum phosphate in WT, Apoe−/− and CRD mice. n=12; r=0.831; R²=0.691;p=0.0008. Dots indicate individual concentration. Error bars indicateSD.

FIGS. 6A and 6B show results of Sortilin 1 co-immunoprecipitation study.FIG. 6A, Sortilin 1 was immunoprecipitated (IP) under lowsalt/stringency conditions from control (CM) or calcified (OM) SMCscultured for 21 days after an IgG-pre-clearance. IgG-IP served ascontrol. One out of two cell donors is shown. Coomassie Blue staining(top) of the gel and Western blot (bottom) of sortilin 1 is shown.Dotted boxes indicate the excised regions of each gel lane, across allfour IP lanes. M; size marker in kDa. Green arrow heads indicate bands(myosin) that disappear in the OM conditions, the red arrow headindicates the molecular weight region corresponding to sortilin 1, andthe black arrow head indicates vimentin. FIG. 6B, The log ratio plot ofthe relative peptide-spectrum matches (PSMs) between OM versus CMconditions.

FIGS. 7A and 7B show establishment of sortilin 1 silencing. SMCs werecultured for 21 days in control medium (CM) and transfected twice perweek during the entire cell culture period of 21 days with siRNA(siSORT1 or scramble control, Scr). FIG. 7A, Sortilin 1 mRNA at day 4,14, and 21. n=2-4. Error bars indicate SD. FIG. 7B, Sortilin 1 protein.b-actin served as loading control. One out of four Western blots isshown.

FIGS. 8A-8E SMCs were cultured for 21 days in control medium (CM) orosteogenic medium (OM) and transfected twice per week during the entirecell culture period of 21 days with siRNA (siSORT1 or scramble control,Scr) or cells were stimulated with recombinant sortilin 1 (rSORT1, 200ng/ml). FIGS. 8A and 8B, Collagen content of the extracellular matrix.FIG. 8C, RUNX2 mRNA expression. n=4 independent cell donor; *p<0.05.FIGS. 8D and 8E, SMCs were stimulated with phosphate (2 mM, 3 mM). 48 hlater sortilin 1 mRNA (D) and protein (E) expression was determined. Oneout of three independent Western blot images is shown. n=3. *p<0.05.Error bars indicate SD.

FIG. 9 shows that phosphate regulating endopeptidase (PHEX) is notdirectly involved in sortilin-1 dependent SMC calcification. SMCs werecultured in osteogenic medium. Sortilin 1 (siSORT1) and PHEX (siPHEX)were silenced by siRNA (50 nM) or scramble control (scr) twice per weekduring the entire cell culture period. Matrix mineralization wasmeasured at day 21. n=3 independent cell donor. Error bars indicate SD.*p<0.05 vs. scr.

FIGS. 10A-10C show that Sortilin 1 does not increase duringosteoblastogenesis. FIG. 10A, Human mesenchymal stromal cells (hMSC)were cultured for up to 21 days in osteogenic medium. Protein expressionwas assessed by Western blot at the indicated time points. FIG. 10B,hMSC were cultured in osteogenic medium to obtain osteoblasts. Sortilin1 (siSORT1) was silenced by siRNA (50 nM) or scramble control (scr)twice per week during the entire cell culture period. FIG. 10C, alkalinephosphatase, tissue non-specific (TNAP) activity measured at day 14. (C)Matrix mineralization measured at day 21. Representative images areshown above. n=3.

FIGS. 11A and 11B show that Sortilin 1 is released within extracellularvesicles from calcified SMC. FIG. 11A, Protein was precipitated fromcell culture supernatant from control SMC (CM, day21); calcified SMC(OM, day 21) or SMC transduced for 7 days with adenovirus sortilin 1(AdSORT1) or AdLacZ as control. Whole cell lysate from control SMC (CM,day21), calcified SMC (OM, day 21) served as control. FIG. 11BExtracellular vesicles were isolated from cell culture supernatant fromcontrol SMC (CM, day21) or calcified SMC (OM, day 21) usingultracentrifugation. 20 μg of protein were loaded on a gale. Whole celllysate from control SMC (CM, day21), calcified SMC (OM, day 21) servedas control.

FIG. 12 shows the association of sortilin 1 to myosin-9 and b-actindisappears in calcified SMC. SMCs were cultured for 21 days in controlmedium (NM) or osteogenic medium (OM). Sortilin 1 was immunoprecipitatedafter an IgG-pre-clearance. IgG-IP served as control. Western blotagainst sortilin 1, myosin-9 (MYH9) and b-actin. Two independent celldonors are shown.

FIGS. 13A and 13B show that association of sortilin 1 to caveolin-1 andtissue non-specific alkaline phosphatase increases in calcified SMC.SMCs were cultured for 21 days in control medium (CM) or osteogenicmedium (OM). Sortilin 1 was immunoprecipitated after anIgG-pre-clearance. IgG-IP served as control. Western blot againstsortilin 1, caveolin-1 (Cav-1) and tissue non-specific alkalinephosphatase (TNAP). FIG. 13A, Western blot of input, sortilin 1 IP andIgG IP. FIG. 13B, four independent cell donors are shown.

FIG. 14 shows that caveolin-1 is induced during osteogenic transition ofhuman vascular SMCs. SMCs were cultured for 21 days in control medium(CM) or osteogenic medium (OM). Western blot at day 1, day 7, and day21. b-actin served as loading control.

FIG. 15 shows that blocking antibody against sortilin 1 reduced alkalinephosphatase (ALP) activity. Calcifying smooth muscle cells (SMCs) werecultured in the presents of a sortilin 1 antibody (10 ug/ml, R&Dsystems) or the corresponding IgG control (10 ug/ml, R&D System) for 14days. Blocking of sortilin 1 using an antibody against the extracellulardomain significantly reduced ALP activity compared to IgG control(p=0.02, n=3).

FIG. 16 shows sortilin 1 propetide reduced ALP activity. Calcifying SMCswere cultured in the presents of the sortilin 1 propeptide (200 ng/ml)for 14 days. Sortilin 1 propeptide significantly reduced ALP activitycompared untreated control (p=0.03, n=3-4).

FIGS. 17A-17E show that sortilin 1 is loaded into matrix vesicles (MV).

FIG. 17A shows transmission electron microscopy-based immunogoldstaining of sortilin 1 on aorta sections of Apoe−/− mice with chronicrenal disease. One out of three animals is shown. Bar=200 nm. FIG. 17Bshows Sortilin 1, Caveolin-1 (Cav-1), and Tissue non-specific alkalinephosphatase (TNAP) expression in MVs isolated from SMCs cultured for 21day in control (CM) or osteogenic (OM) medium. One out of threeexperiments is shown. FIG. 17C shows identification of sortilin 1peptides in MVs using mass spectrometry. Peptide sequences are, from topto bottom: AAAAGGAFPR (SEQ ID NO: 2); TEFGMAIGPENSGK (SEQ ID NO: 3);SAPGEDEECG (SEQ ID NO: 4); and CTSNFLSPEK (SEQ ID NO: 5). FIG. 17D, SMCswere cultured for 14, and 21 days in control (CM) or osteogenic medium(OM) under the adenoviral overexpression of sortilin 1 (Ad-SORT1) or thecontrol vector (Ad-LacZ). MVs were isolated from the SMCs supernatant.Top: Western blot. Numbers represents the number of sequensed peptidesand the corresponding peptide spectral matches (PSM). FIG. 17D shows MVrelease. n=4 independent cell donor. *p<0.05. Error bars indicate SD

FIGS. 18A-18H show that sortilin 1 re-distributes to lipidraft/caveolae-enriched membrane in calcified SMCs. FIG. 18A, SMCs werecultured for 21 days in control (CM) or osteogenic (OM) medium. Lipidraft/caveolae-enriched membrane (CEM) were resolved from other cellularconstituents (nCEM) by a hydrodynamic method. Caveolin-1 (Cav-1) andsortilin 1 protein expression were assessed by Western blot. One out of3 independent cell donors is shown. FIGS. 18B and 18C, SMCs werecultured for 21 days in control medium (CM), or osteogenic medium (OM).Sortilin 1 and Caveolin-1 were silenced by siRNA (siSORT, siCav-1 orscramble control, Scr) twice per week during the entire cell cultureperiod. Western blot of whole cell lysate (FIGS. 18B and 18C) andCEM/nCEM (FIG. 18C). One out of 3 independent cell donors is shown.FIGS. 18D and 18E, SMCs were cultured for 14 (FIG. 18D, Tissuenon-specific alkaline phosphatase (TNAP) activity) or 21 (FIG. 18E,matrix mineralization) days in control medium (CM), or osteogenic medium(OM). Caveolin-1 was silenced by siRNA (siCav-1 or scramble control,Scr) twice per week during the entire cell culture period. n=4independent cell donors. *p<0.05. FIGS. 18F and 18G, SMCs were isolatedfrom caveolin-1-deficient mice (Cav-1^(−/−)) or wild type mice (WT) andcultured for 14 (FIG. 18F, Tissue non-specific alkaline phosphatase(TNAP) activity) or 21 (FIG. 18G, matrix mineralization) days inosteogenic medium with and without mouse recombinant sortilin 1 (rSORT1,200 ng/ml). n=4. *p<0.05. FIG. 18H shows TNAP activity in MVs isolatedfrom SMCs cultured for 14 days in control (CM) or osteogenic (OM)medium. Sortilin 1 was silenced by siRNA (siSORT, or scramble control,Scr) twice per week during the entire cell culture period. n=4independent cell donor. *p<0.05. Error bars indicate SD.

FIG. 19 shows that phosphorylation on S₈₂₅ and S₈₁₉ affect cellular ALPactivity and matrix mineralization.

DETAILED DESCRIPTION

In one aspect provided herein is a method of decreasing, inhibiting orpreventing calcification of SMCs. Generally, the method comprisesinhibiting sortilin 1. In some embodiments, the method comprisingcontacting a compound with SMCs, wherein the compound decreases,inhibits, prevents or reduces: (i) activity or amount of sortilin 1 inSMCs; (ii) expression of a nucleic acid encoding sortilin 1 in SMCs; or(iii) phosphorylation of serine 819 or/and 825.

The compounds which can decrease, inhibit, prevent or reduce theactivity of sortilin or expression thereof are also referred to assortilin inhibitors herein.

The terms “decrease,” “inhibit,” “reduced,” or “reduction,” in referenceto activity, amount or expression of a nucleic acid encoding sortilin 1generally mean a decrease by a statistically significant amount.However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or“inhibit” means a decrease by at least 10% as compared to a referencelevel, for example a decrease by at least about 15%, or at least about20%, or at least about 25%, or at least about 30%, or at least about35%, or at least about 40%, or at least about 45%, or at least about50%, or at least about 55%, or at least about 60%, or at least about65%, or at least about 70%, or at least about 75%, or at least about80%, or at least about 85%, or at least about 90%, or at least about 95%but not 100% (e.g. absent level as compared to a reference sample)decrease. In some embodiments, decrease can be 100% (e.g., a level belowlimit of detection). Reference level can be the level in absence of theinhibitor.

Methods disclosed herein can be used to prevent or treat atheroscleroticcalcification, medial calcification, aortic valve calcification, andother conditions characterized by cardiovascular calcification. Forexample, the methods disclosed herein can be used to prevent or treatcalcification in patients with any mineral imbalance disorders,including severe renal failure on hemodialysis, hemodialysis AVgrafts/shunts, vein grafts, various vascular anastomosis, diabetes,Paget's disease, rheumatoid arthritis, osteoporosis or some forms ofosteoarthritis. Without wishing to be bound by a theory, in theseindications, progression of vascular or valvular calcification candevelop within weeks-months. In some embodiments, vascular/valvular(e.g., aortic valve, mitral annulus) calcification can be associatedwith chronic renal insufficiency or end-stage renal disease. In someembodiments, vascular/valvular calcification can be associated with pre-or post-dialysis or uremia. In some embodiments, vascular/valvularcalcification can be associated with diabetes type I or II. In someembodiments, vascular/valvular calcification can be associated with acardiovascular disorder. In some embodiments, vascular calcification canbe associated with bone disease (Paget's disease, rheumatoid arthritis,osteoporosis, osteoarthritis).

Accordingly, presented herein are also methods for decreasing,inhibiting, preventing, or treating calcification, e.g., cardiovascularcalcification, in a subject. The method comprising inhibiting theactivity, amount or expression level of sortilin 1 in the subject. Insome embodiments, the method comprising administering to a subject inneed thereof a compound, wherein the compound decreases, inhibits, orreduces:

(i) activity or amount of sortilin 1 in SMCs;

(ii) expression of a nucleic acid encoding sortilin 1 in SMCs; or

(iii) phosphorylation of serine 819 or/and 825.

In some embodiments, the inhibitor retards or reverses the formation,nucleation, aggregation, growth or deposition of extracellular matrixhydroxyapatite crystal deposits. In some embodiments, the inhibitorprevents the formation, nucleation, growth or deposition ofextracellular matrix hydroxyapatite crystal deposits. In someembodiments, the inhibitor prevents the release, nucleation andcalcification of matrix vesicles.

Without wishing to be bound by a theory, the inhibitor can act directlyon sortilin 1 itself or a molecule or enzyme or on a pathway that actson sortilin 1. For example, as the work reported herein demonstrates,sortilin 1 is a target for microRNA 125b (mir-125b). Thus, compounds orcompositions that can increase miR-125b levels can repress expression ofSORT 1 since miR-125b binds to 3′UTR of SORT1 and repress itsexpression.

The inventors have discovered that silencing of sortilin 1 significantlyreduced alkaline phosphatase activity, matrix mineralization, matrixvesicle release, and increased calcific potential of such vesicles.Accordingly, in some embodiments, the sortilin inhibitor inhibits orreduces tissue non-specific alkaline phosphatase activity (TNAP) by atleast 10%, for example a decrease by at least about 15%, or at leastabout 20%, or at least about 25%, or at least about 30%, or at leastabout 35%, or at least about 40%, or at least about 45%, or at leastabout 50%, or at least about 55%, or at least about 60%, or at leastabout 65%, or at least about 70%, or at least about 75%, or at leastabout 80%, or at least about 85%, or at least about 90%, or at leastabout 95% but not 100% (e.g. absent level as compared to a referencesample) decrease. In some embodiments, decrease can be 100% (e.g., alevel below limit of detection). Reference level can be the level inabsence of the inhibitor.

The inventors have shown that sortilin 1 associates with TNAP andCaveolin-1. Accordingly, in some embodiments, the sortilin inhibitor caninhibit the association of sortilin 1 to TNAP and/or Caveolin-1. In someembodiments, the sortilin inhibitor decreases, inhibits, prevents orreduces association of sortilin 1 to TNAP and/or Caveolin-1 by at least10%, for example a decrease by at least about 15%, or at least about20%, or at least about 25%, or at least about 30%, or at least about35%, or at least about 40%, or at least about 45%, or at least about50%, or at least about 55%, or at least about 60%, or at least about65%, or at least about 70%, or at least about 75%, or at least about80%, or at least about 85%, or at least about 90%, or at least about 95%but not 100% (e.g. absent level as compared to a reference sample)decrease. In some embodiments, decrease can be 100% (e.g., a level belowlimit of detection). Reference level can be the level in absence of theinhibitor.

In some embodiments, the sortilin inhibitor can decrease matrixmineralization by at least 10%, for example a decrease by at least about15%, or at least about 20%, or at least about 25%, or at least about30%, or at least about 35%, or at least about 40%, or at least about45%, or at least about 50%, or at least about 55%, or at least about60%, or at least about 65%, or at least about 70%, or at least about75%, or at least about 80%, or at least about 85%, or at least about90%, or at least about 95% but not 100% (e.g. absent level as comparedto a reference sample) decrease. In some embodiments, decrease can be100% (e.g., a level below limit of detection). Reference level can bethe level in absence of the inhibitor.

Inventors have also discovered that there is a significant inversecorrelation between sortilin 1 and phosphate regulating endopeptidase(PHEX). Inhibition of sortilin 1 increased PHEX expression levels. Thus,in some embodiments, the sortilin inhibitor can increase PHEX expressionby at least 10%, for example an increase by at least about 15%, or atleast about 20%, or at least about 25%, or at least about 30%, or atleast about 35%, or at least about 40%, or at least about 45%, or atleast about 50%, or at least about 55%, or at least about 60%, or atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, at leastabout 95%, at least 1-fold, at least 1.25-folds, at least 2-fold, atleast 2.5-folds, at least 5-folds, at least 10-folds, at least 50-foldsor more. Reference level can be the level in absence of the inhibitorReference level can be the level in absence of the inhibitor.

Based on their work, the inventors have further discovered that sortilin1 is a target for microRNA 125b (mir125b). Mir125b binds to 3′UTR ofSORT1 and represses its expression. Thus, without wishing to be bound bya theory, compounds or compositions that increase expression of mir125bcan be used to inhibit sortilin 1 by repressing the expression ofSORT 1. Accordingly, in some embodiments, the sortilin inhibitor canincrease expression level of microRNA 125b by at least 10%, for examplean increase by at least about 15%, or at least about 20%, or at leastabout 25%, or at least about 30%, or at least about 35%, or at leastabout 40%, or at least about 45%, or at least about 50%, or at leastabout 55%, or at least about 60%, or at least about 65%, or at leastabout 70%, or at least about 75%, or at least about 80%, or at leastabout 85%, or at least about 90%, at least about 95%, at least 1-fold,at least 1.25-folds, at least 2-fold, at least 2.5-folds, at least5-folds, at least 10-folds, at least 50-folds or more. Reference levelcan be the level in absence of the inhibitor.

Without limitations, the inhibitor, can be selected from the groupconsisting of small organic or inorganic molecules; saccharines;oligosaccharides; polysaccharides; biological macromolecules, e.g.,peptides, proteins, and peptide analogs and derivatives;peptidomimetics; antibodies and antigen binding fragments thereof;nucleic acids; nucleic acid analogs and derivatives; an extract madefrom biological materials such as bacteria, plants, fungi, or animalcells; animal tissues; naturally occurring or synthetic compositions;and any combinations thereof.

In some embodiments, the inhibitor is a small molecule. As used herein,the term “small molecule” can refer to compounds that are “naturalproduct-like,” however, the term “small molecule” is not limited to“natural product-like” compounds. Rather, a small molecule is typicallycharacterized in that it contains several carbon-carbon bonds, and has amolecular weight of less than 5000 Daltons (5 kDa), preferably less than3 kDa, still more preferably less than 2 kDa, and most preferably lessthan 1 kDa. In some cases it is preferred that a small molecule have amolecular weight equal to or less than 700 Daltons.

In some embodiments, the inhibitor is a nucleic acid molecule or ananalog or derivate thereof. As used herein, the term “nucleic acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides, including analogs or derivatives thereof that arecovalently linked together. Exemplary oligonucleotides include, but arenot limited to, single-stranded and double-stranded siRNAs and other RNAinterference reagents (RNAi agents or iRNA agents), shRNA (short hairpinRNAs), antisense oligonucleotides, aptamers, ribozymes, and microRNAs(miRNAs). The nucleic acids can be single stranded or double stranded.The nucleic acid can be DNA, RNA or a hybrid, where the nucleic acidcontains any combination of deoxyribo- and ribonucleotides, and anycombination of uracil, adenine, thymine, cytosine and guanine. Thenucleic acids can comprise one or more backbone modifications, e.g.,phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970)),phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), or peptide nucleic acid linkages (see Egholm,J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); and Nielsen, Nature, 365:566 (1993), content of all ofwhich is herein incorporated by reference. The nucleic acids can alsoinclude modifications to nucleobase and/or sugar moieties ofnucleotides. Exemplary sugar modifications at the sugar moiety includereplacement of 2′-OH with halogens (e.g., fluoro), O-methyl,O-methoxyethyl, NH₂, SH and S-methyl. In some embodiments, the nucleicacid is a peptide nucleic acid (PNA). Without wishing to be bound by atheory, nucleic acid inhibitors can decrease, inhibit, or reduce theexpression or amount of the nucleic acid encoding a component of thecomplex. Computational and experimental methods, including highthroughput screening assays, for producing nucleic acid inhibitors,e.g., antisense oligonucleotides, siRNAs, ribozymes, aptamers, and thelike, targeted to any target sequence are known in the art and availableto one of skill in the art.

In some embodiments, the inhibitor is short interfering RNA (siRNA). Theterm “short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. A siRNA can be chemicallysynthesized, it can be produced by in vitro transcription, or it can beproduced within a host cell. siRNA molecules can also be generated bycleavage of double stranded RNA, where one strand is identical to themessage to be inactivated. The term “siRNA” refers to small inhibitoryRNA duplexes that induce the RNA interference (RNAi) pathway. Thesemolecules can vary in length (generally 18-30 base pairs) and containvarying degrees of complementarity to their target mRNA in the antisensestrand. Some, but not all, siRNA have unpaired overhanging bases on the5′ or 3′ end of the sense strand and/or the antisense strand. The term“siRNA” includes duplexes of two separate strands, as well as singlestrands that can form hairpin structures comprising a duplex region.

In some embodiments, the inhibitor is an antisense oligonucleotide orsiRNA molecule comprising a part of (e.g., 10-50, 12-40, 15-30, 16-25,or 18-22 consecutive nucleotides) of the antisense sequence of a nucleicacid encoding sortilin 1, e.g. SORT 1. Nucleic acid sequence of humanSORT 1 can be accessed by NCBI Reference Sequence: NM_002959.4. In someembodiments, the nucleic acid encoding sortilin 1 is SORT 1 mRNA.

In some embodiments, the inhibitor comprises the nucleic acid sequence

(SEQ ID NO: 6) 5′ -GAAUUUGGCAUGGCUAUUG -3′; (SEQ ID NO: 7) 5′-GAAGGACUAUACCAUAUGG -3′; (SEQ ID NO: 8) 5′ -GAGCUAGGUCCAUGAAUAU -3′; or(SEQ ID NO: 9) 5′ -GAGACUAUGUUGUGACCAA-3′.

In some embodiments, the inhibitor is an antibody or a fragment thereof.The terms “antibody” and “antibodies” include polyclonal antibodies,monoclonal antibodies, humanized or chimeric antibodies, single chain Fvantibody fragments, Fab fragments, and F(ab)₂ fragments. Antibodieshaving specific binding affinity for sortilin 1 can be produced throughstandard methods. Alternatively, antibodies may be commerciallyavailable, for example, from R&D Systems, Inc., Minneapolis, Minn.

As used herein, the terms “antibody” and “antibodies” refer to intactantibody, or a binding fragment thereof that competes with the intactantibody for specific binding and includes chimeric, humanized, fullyhuman, and bispecific antibodies. In some embodiments, binding fragmentsare produced by recombinant DNA techniques. In additional embodiments,binding fragments are produced by enzymatic or chemical cleavage ofintact antibodies. Binding fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies.

Unless it is specifically noted, as used herein a “fragment thereof” inreference to an antibody refers to an immunespecific fragment, i.e., anantigen-specific or binding fragment.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular epitope contained within an antigen, can be preparedusing standard hybridoma technology. In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described by Kohler, G. et al., Nature, 1975, 256:495, the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 1983, 4:72;Cole et al., Proc. Natl. Acad. Sci. USA, 1983, 80:2026), and theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., 1983, pp. 77-96). Such antibodies can be ofany immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. The hybridoma producing the monoclonal antibodies ofthe invention can be cultivated in vitro or in vivo.

Polyclonal antibodies are heterogeneous populations of antibodymolecules that are specific for a particular antigen, which arecontained in the sera of the immunized animals. Polyclonal antibodiesare produced using well-known methods. A chimeric antibody is a moleculein which different portions are derived from different animal species,such as those having a variable region derived from a murine monoclonalantibody and a human immunoglobulin constant region. Chimeric antibodiescan be produced through standard techniques. Antibody fragments thathave specific binding affinity for a component of the complex can begenerated by known techniques. For example, such fragments include, butare not limited to, F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed. See, forexample, Huse et al., 1989, Science, 246: 1275. Single chain Fv antibodyfragments are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge (e.g., 15 to 18 amino acids),resulting in a single chain polypeptide. Single chain Fv antibodyfragments can be produced through standard techniques. See, for example,U.S. Pat. No. 4,946,778.

In some embodiments, the antibody or antigen-binding fragment thereof ismurine. In some embodiments, the antibody or antigen-binding fragmentthereof is from rabbit. In some embodiments, the antibody orantigen-binding fragment thereof is from rat. In other embodiments, theantibody or antigen binding fragment thereof is human. In someembodiments the antibody or antigen-binding fragment thereof isrecombinant, engineered, humanized and/or chimeric.

In some embodiments, an antibody, or antigen binding fragment, variant,or derivative thereof for use in the methods of the invention bindsspecifically to at least one epitope of target molecule (e.g., sortilin1), i.e., binds to such an epitope more readily than it would bind to anunrelated, or random epitope; binds preferentially to at least oneepitope of sortilin, i.e., binds to such an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope;competitively inhibits binding of a reference antibody which itselfbinds specifically or preferentially to an epitope of the targetmolecule (e.g., sortilin 1); or binds to at least one epitope of thetarget molecule (e.g., sortilin 1) with an affinity characterized by adissociation constant Kd of about 5×10⁻² M, about 10⁻² M, about 5×10⁻³M, about 10⁻³M, about 5×10⁻⁴M, about 10⁻⁴ M, about 5×10⁻⁵M, about 10⁻⁵M,about 5×10⁻⁶ M, about 10⁻⁶ M, about 5×10⁻⁷M, about 10⁻⁷M, about 5×10⁻⁸M,about 10⁻⁸ M, about 5×10⁻⁹ M, about 10⁻⁹M, about 5×10⁻¹⁰ M, about 10⁻¹⁰M, about 5×10⁻¹¹ M, about 10⁻¹¹M, about 5×10⁻¹²M, about 10⁻¹² M, about5×10⁻¹³M, about 10⁻¹³M, about 5×10⁻¹⁴M, about 10⁻¹⁴M, about 5×10⁻¹⁵M, orabout 10⁻¹⁵M.

In some embodiments, the antibody or fragment thereof preferentiallybinds to a human sortilin polypeptide or fragment thereof, relative to amurine sortilin polypeptide or fragment thereof.

As used in the context of antibody binding dissociation constants, theterm “about” allows for the degree of variation inherent in the methodsutilized for measuring antibody affinity. For example, depending on thelevel of precision of the instrumentation used, standard error based onthe number of samples measured, and rounding error, the term “about 10⁻²M” can include, for example, from 0.05 M to 0.005 M.

In some embodiments, an antibody, or antigen binding fragment, variant,or derivative thereof for use in the methods described herein bindssortilin polypeptides or fragments or variants thereof with an off rate(k(off)) of less than or equal to about 5×10⁻² sec⁻¹, about 10⁻² sec⁻¹,about 5×10⁻³ sec⁻¹, about 10⁻³ sec⁻¹, about 5×10⁻⁴ sec⁻¹, about 10⁻⁴sec⁻¹, about 5×10⁻⁴ sec⁻¹, about 10⁻⁴ sec⁻¹, about 5×10⁻⁵ sec⁻¹, about10⁻⁵ sec⁻¹, about 5×10⁻⁶ sec⁻¹, about 10⁻⁶ sec⁻¹, about 5×10⁻⁷ sec⁻¹, orabout 10⁻⁷ sec⁻¹.

In other embodiments, an antibody, or antigen-binding fragment, variant,or derivative thereof for use in the methods described herein bindssortilin polypeptides or fragments or variants thereof with an on rate(k(on)) of greater than or equal to about 10³M⁻¹ sec⁻¹, about 5×10³M⁻¹sec⁻¹, 10⁴M⁻¹ sec⁻¹, about 5×10⁴M⁻¹ sec⁻¹, 10⁵M⁻¹ sec⁻¹, about 5×10⁵M⁻¹sec⁻¹, 10⁶M⁻¹ sec⁻¹, about 5×10⁶ M⁻¹ sec⁻¹, 10⁷M⁻¹ sec⁻¹, or about5×10⁷M⁻¹ sec⁻¹.

The binding affinity and dissociation rate of an antibody for use in themethods described herein can be determined by any method known in theart. For example, the binding affinity can be measured by competitiveELISAs, RIAs, BIACORE™, or KINEXA™ technology. The dissociation ratealso can be measured by BIACORE™ or KINEXA™ technology. The bindingaffinity and dissociation rate are measured by surface plasmon resonanceusing, e.g., a BIACORE™.

In some embodiments, an antibody or an antigen-binding fragment for usein the methods described herein modulates the binding of a secondmolecule to sortilin. In some embodiments, the modulation is enhancementof the binding of the second molecule to sortilin. In some embodiments,the modulation is inhibition of the binding of the second molecule tosortilin. The IC50 of such inhibition can be measured by any methodknown in the art, e.g., by ELISA, RIA, or Functional Antagonism. In someembodiments, the IC50 is between 0.1 and 500 nM. In some embodiments,the IC50 is between 10 and 400 nM. In yet other embodiments, theantibody or portion thereof has an IC50 of between 60 nM and 400 nM.

Antibodies for use in the methods of the invention can be generated byimmunization of a suitable host (e.g., vertebrates, including humans,mice, rats, sheep, goats, pigs, cattle, horses, reptiles, fishes,amphibians, and in eggs of birds, reptiles and fish). Such antibodiesmay be polyclonal or monoclonal. In some embodiments, the host isimmunized with an immunogenic sortilin. In other embodiments, the hostis immunized with sortilin associated with a cell membrane of an intactor disrupted cell and antibodies for use in the methods of the inventionare identified by binding to sortilin.

In some embodiments, the sortilin antigen is administered with anadjuvant to stimulate the immune response. Adjuvants often need to beadministered in addition to antigen in order to elicit an immuneresponse to the antigen. These adjuvants are usually insoluble ornondegradable substances that promote nonspecific inflammation, withrecruitment of mononuclear phagocytes at the site of immunization.Examples of adjuvants include, but are not limited to, Freund'sadjuvant, RIBI (muramyl dipeptides), ISCOM (immunostimulating complexes)or fragments thereof.

For a review of methods for making antibodies, see, e.g., Harlow andLane, Antibodies, A Laboratory Manual (1988); Yelton, D. E. et al., Ann.Rev. of Biochem. 50:657-80. (1981); and Ausubel et al., CurrentProtocols in Molecular Biology (New York: John Wiley & Sons) (1989).Determination of immunoreactivity with an immunogenic sortilinpolypeptide may be made by any of several methods well known in the art,including, e.g., immunoblot assay and ELISA.

Anti-sortilin antibodies for use in the methods described herein can beof any isotype. An antibody of any desired isotype can be produced byclass switching. For class switching, nucleic acids encoding VL or VH,that do not include any nucleotide sequences encoding CL or CH, areisolated using methods well known in the art. The nucleic acids encodingVL or VH are then operatively linked to a nucleotide sequence encoding aCL or CH from a desired class of immunoglobulin molecule. This can beachieved using a vector or nucleic acid that comprises a CL or CH chain,as described above. For example, an anti-sortilin antibody for use inthe methods described herein that was originally IgM can be classswitched to an IgG. Further, the class switching can be used to convertone IgG subclass to another, e.g., from IgG1 to IgG2.

The class and subclass of anti-sortilin antibodies can be determined byany method known in the art. In general, the class and subclass of anantibody can be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies are availablecommercially. The class and subclass can be determined by ELISA, WesternBlot, as well as other techniques. Alternatively, the class and subclasscan be determined by sequencing all or a portion of the constant domainsof the heavy and/or light chains of the antibodies, comparing theiramino acid sequences to the known amino acid sequences of various classand subclasses of immunoglobulins, and determining the class andsubclass of the antibodies.

In certain embodiments both the variable and constant regions ofsortilin antibodies or immunospecific fragments thereof for use in thetreatment methods disclosed herein are fully human. Fully humanantibodies can be made using techniques that are known in the art and asdescribed herein. For example, fully human antibodies against a specificantigen can be prepared by administering the antigen to a transgenicanimal which has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled.Exemplary techniques that can be used to make such antibodies aredescribed in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Othertechniques are known in the art. Fully human antibodies can likewise beproduced by various display technologies, e.g., phage display or otherviral display systems, as described in more detail elsewhere herein.

Antibodies or fragments thereof for use in the treatment methodsdisclosed herein include derivatives that are modified, e.g., by thecovalent attachment of any type of molecule to the antibody such thatcovalent attachment does not prevent the antibody from specificallybinding to its cognate epitope. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, or derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein. Any of numerous chemical modifications may becarried out by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, etc. . . . . Additionally,the derivative can contain one or more non-classical amino acids.

In some embodiments, antibody or fragment thereof for use in the methodsdisclosed herein will not elicit a deleterious immune response in themammal to be treated, e.g., in a human. In one embodiment, theantibodies or fragments thereof for use in the methods disclosed hereincan be modified to reduce their immunogenicity using art-recognizedtechniques. For example, antibodies can be humanized, primatized,deimmunized, or chimeric antibodies can be made. These types ofantibodies are derived from a nonhuman antibody, typically a murine orprimate antibody, that retains or substantially retains theantigen-binding properties of the parent antibody, but which is lessimmunogenic in humans. This can be achieved by various methods,including (a) grafting the entire non-human variable domains onto humanconstant regions to generate chimeric antibodies; (b) grafting at leasta part of one or more of the non-human complementarity determiningregions (CDRs) into a human framework and constant regions with orwithout retention of critical framework residues; or (c) transplantingthe entire non-human variable domains, but “cloaking” them with ahuman-like section by replacement of surface residues. Such methods aredisclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen etal., Science 239:15341536 (1988); Padlan, Molec. Immun. 28:489-498(1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762; and 6,190,370, all of which are herebyincorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes (see, e.g., WO9852976A1,WO0034317A2). For example, VH and VL sequences from the startingantibody are analyzed and a human T cell epitope “map” from each Vregion showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative VH and VL sequences are designed comprising combinations ofamino acid substitutions and these sequences are subsequentlyincorporated into a range of binding polypeptides, e.g., anti-sortilinantibodies or immunospecific fragments thereof for use in the methodsdisclosed herein, which are then tested for function. Typically, between12 and 24 variant antibodies are generated and tested. Complete heavyand light chain genes comprising modified V and human C regions are thencloned into expression vectors and the subsequent plasmids introducedinto cell lines for the production of whole antibody. The antibodies arethen compared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences. See, forexample, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered nonfunctional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected nonhuman monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903(1988)). See also, U.S. Pat. No. 5,565,332. [0162] In anotherembodiment, DNA encoding desired monoclonal antibodies may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The isolatedand subcloned hybridoma cells serve as a preferred source of such DNA.Once isolated, the DNA may be placed into expression vectors, which arethen transfected into prokaryotic or eukaryotic host cells such as E.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells ormyeloma cells that do not otherwise produce immunoglobulins. Moreparticularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

Anti-sortilin antibodies can be purchased from Abcam (Cambridge, Mass.,USA) or R&D Systems (Minneapolis, Minn., USA). Further, methods foridentification and design of ligands capable of binding specifically toSortilin 1 are described, for example, in US Patent ApplicationPublication No. 2011/1060439, content of which is incorporated herein byreference.

The cell, e.g. SMC, can be contacted with the inhibitor in a cellculture e.g., in vitro or ex vivo, or the inhibitor can be administratedto a subject, e.g., in vivo. In some embodiments, the inhibitor can beadministrated to a subject to decrease, inhibit, prevent, reduce, and/ortreat calcification. In some embodiments, the cell is an interstitialvalvular cell. In some embodiments, the cell is an osteoblast. In someembodiments, the cell is an osteoclast. In some embodiments, the cell isa mesenchymal stem cell. In some embodiments, the cell is an endothelialcell. In some embodiments, the cell is a macrophage. In someembodiments, the cell is a monocyte. In some embodiments, the cell is adendritic cell. In some embodiments, the cell is a lymphocyte.

The term “contacting” or “contact” as used herein in connection withcontacting a cell includes subjecting the cells to an appropriateculture media, which comprises the indicated inhibitor. Where the cellis in vivo, “contacting” or “contact” includes administering theinhibitor in a pharmaceutical composition to a subject via anappropriate administration route such that the inhibitor contacts thecell in vivo.

As described herein, the inhibitors can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art.

The term “ex vivo” refers to cells which are removed from a livingorganism and cultured outside the organism (e.g., in a test tube). If exvivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells viamethods known or available to one of skill in the art. For example, thecells can be kept in a culture and inhibitor can be added to the culturemedia. The treated cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

Generally, any amount of the compound can be contacted with the SMC. Insome embodiments, compound is contacted at a concentration in the rangeof from about 0.1 nM to about 1000 mM. Preferably the compound iscontacted in the range of from about 0.1 μM to about 10 μM.

Additionally, the compound can be contacted with the SMC for asufficient time to allow the compound to be taken up by the SMC andinteract with its target. For a non-limiting example, the compound canbe contacted with the SMC at least 15 minutes before assaying foractivity or amount of sortilin 1 or assaying for the amount orexpression of the nucleic acid encoding sortilin 1.

For administration to a subject, the inhibitor can be formulated inpharmaceutically acceptable compositions which comprise the inhibitorformulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. The inhibitors can be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), lozenges,dragees, capsules, pills, tablets (e.g., those targeted for buccal,sublingual, and systemic absorption), boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally; (9) nasally; or (10) local administration (e.g., drugeluting stent, pluronic gel). Additionally, the inhibitors can beimplanted into a patient or injected using a drug delivery composition.See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 35 3,270,960.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (24) C2-C12 alcohols, such as ethanol; (26) lipidnanoparticles; and (27) other non-toxic compatible substances employedin pharmaceutical formulations. The carrier or excipient can includetime delay material, such as glyceryl monostearate or glyceryldistearate alone or with a wax, or other materials well known in theart. Wetting agents, coloring agents, release agents, coating agents,sweetening agents, flavoring agents, perfuming agents, preservative andantioxidants can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

Pharmaceutically-acceptable antioxidants include, but are not limitedto, (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lectithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acids, and the like.

The preparation of pharmaceutical compositions is well known in the artand has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack PublishingCo., Easton, Pa., 1990, content of which of which is incorporated hereinby reference in its entirety.

The pharmaceutical compositions can be made up in a solid form(including granules, powders or suppositories) or in a liquid form(e.g., solutions, suspensions, or emulsions). The pharmaceuticalcompositions can be subjected to conventional pharmaceutical operationssuch as sterilization and/or can contain conventional adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifiers, buffers etc.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound can be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms can also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms can also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting, sweetening,flavoring, and perfuming agents.

For purposes of parenteral administration, solutions in sesame or peanutoil or in aqueous propylene glycol can be employed, as well as sterileaqueous solutions of the corresponding water-soluble salts. Such aqueoussolutions can be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. These aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal injection purposes. In this connection,the sterile aqueous media employed are all readily obtainable bystandard techniques well-known to those skilled in the art methods ofpreparing various pharmaceutical compositions with a certain amount ofactive ingredient are known, or will be apparent in light of thisdisclosure, to those skilled in this art.

The inhibitors can also be administered in controlled releaseformulations such as a slow release or a fast release formulation. Suchcontrolled release formulations of the combination of this invention maybe prepared using methods well known to those skilled in the art. Themethod of administration will be determined by the attendant physicianor other person skilled in the art after an evaluation of the subject'sconditions and requirements.

The amount of inhibitor that can be combined with a carrier material toproduce a single dosage form will generally be that amount of theinhibitor that produces a therapeutic effect. Generally out of onehundred percent, this amount will range from about 0.01% to 99% ofinhibitor. In some embodiment, amount of the inhibitor in thecomposition can be selected from the range from about 0.1% to about 99%(w/w), from about 1% to about 90% (w/w), from about 2% to about 80%(w/w), from about 5% to about 75% (w/w), from about 5% to about 50%(w/w), from about 10% (w/w) to about 60% (w/w), from about 0.01% toabout 95% (w/v), from about 0.1% to about 90% (w/w), from about 1% toabout 85% (w/w), from about 10% to about 50% (w/w), from about 1% toabout 99% (w/w), from about 0.05% to about 99% (w/w), from about 0.1% toabout 90% (w/w), from about 0.5% to about 85% (w/w), or from about 5% toabout 80% (w/w) of the total composition.

In some embodiments, the composition comprises a therapeuticallyeffective amount of the complex inhibitor for the treatment orprevention of cardiovascular calcification.

As used herein, the term “therapeutically effective amount” means anamount of the therapeutic agent which is effective to provide a desiredoutcome. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art. Generally, atherapeutically effective amount can vary with the subject's history,age, condition, sex, as well as the severity and type of the medicalcondition in the subject, and administration of other agents thatinhibit pathological processes in neurodegenerative disorders.

Furthermore, therapeutically effective amounts will vary, as recognizedby those skilled in the art, depending on the specific disease treated,the route of administration, the excipient selected, and the possibilityof combination therapy. In some embodiments, the therapeuticallyeffective amount can be in a range between the ED₅₀ and LD₅₀ (a dose ofa therapeutic agent at which about 50% of subjects taking it arekilled). In some embodiments, the therapeutically effective amount canbe in a range between the ED₅₀ (a dose of a therapeutic agent at which atherapeutic effect is detected in at least about 50% of subjects takingit) and the TD₅₀ (a dose at which toxicity occurs at about 50% of thecases). Guidance regarding the efficacy and dosage which will deliver atherapeutically effective amount of a compound can be obtained fromanimal models of condition to be treated.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compositions that exhibit large therapeutic indices are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay. Examples of suitable bioassays include DNA replication assays,transcription based assays, and immunological assays.

The dosage can be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the complexinhibitors are administered so that the inhibitor is given at a dosefrom 1 μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to100 mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg,1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood thatranges given here include all intermediate ranges, for example, therange 1 mg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to10 mg/kg, and the like. It is to be further understood that the rangesintermediate to the given above are also within the scope of thisinvention, for example, in the range 1 mg/kg to 10 mg/kg, dose rangessuch as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg, andthe like. For protein based inhibitors (such as antibodies) onepreferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10mg/kg to 20 mg/kg).

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the pharmaceutically active agent at adesired site. The inhibitors can be administered in any suitable manner.The manner of administration can be chosen based on, for example,whether local or systemic treatment is desired, and on the area to betreated. Accordingly, a composition can be administered by anyappropriate route which results in effective treatment in the subject,i.e., administration results in delivery to a desired location in thesubject where at least a portion of the pharmaceutically active agent isdelivered. Exemplary modes of administration include, but are notlimited to, implant, injection, infusion, instillation, implantation, oringestion. “Injection” includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion.

In some embodiments, a composition described herein can be implanted ina subject. As used herein, the term “implanted,” and grammaticallyrelated terms, refers to the positioning of the composition in aparticular locus in the subject, either temporarily, semi-permanently,or permanently. The term does not require a permanent fixation of thecomposition in a particular position or location.

With respect to duration and frequency of administration or treatment,it is typical for skilled clinicians to monitor subjects in order todetermine when the treatment is providing therapeutic benefit, and todetermine whether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment ormake other alteration to treatment regimen. The dosing schedule can varyfrom once a week to daily depending on a number of clinical factors,such as the subject's sensitivity to the polypeptides. The desired dosecan be administered at one time or divided into subdoses, e.g., 2-4subdoses and administered over a period of time, e.g., at appropriateintervals through the day or other appropriate schedule. Such sub-dosescan be administered as unit dosage forms. In some embodiments,administration is chronic, e.g., one or more doses daily over a periodof weeks or months. Examples of dosing schedules are administration oncea month, once every two week, once a week, once every other day, daily,twice daily, three times daily or four or more times daily over a periodof 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4months, 5 months, or 6 months or more.

In some embodiments, the inhibitor can be co-administered to the subjectwith in combination with a pharmaceutically active agent or therapeuticagent. Without limitations, the inhibitor can be administered before,concurrently, or after administration of the therapeutic agent. Thus, asused herein, the term “co-administer” refers to administration of two ormore agents (e.g., the inhibitor and the pharmaceutically active agent)within a 24 hour period of each other, for example, as part of aclinical treatment regimen. In other embodiments, “co-administer” refersto administration within 12 hours, within 6 hours, within 5 hours,within 4 hours, within 3 hours, within 2 hours, within 1 hour, within45, within 30 minutes, within 20, within 15 minutes, within 10 minutes,or within 5 minutes of each other. In other embodiments, “co-administer”refers to administration at the same time, either as part of a singleformulation or as multiple formulations that are administered by thesame or different routes. When the inhibitor and the pharmaceuticallyactive agent are administered in different pharmaceutical compositionsor at different times, routes of administration can be same ordifferent.

Exemplary pharmaceutically active compound include, but are not limitedto, those found in Harrison's Principles of Internal Medicine, 13^(th)Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians'Desk Reference, 50^(th) Edition, 1997, Oradell N.J., Medical EconomicsCo.; Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete contents of all of which are incorporated herein byreference.

In some embodiments, pharmaceutically active agent can include thoseagents known in the art for treating cardiovascular calcification, suchas vitamin D sterols and/or RENAGEL®. Vitamin D sterols can includecalcitriol, alfacalcidol, doxercalciferol, maxacalcitol or paricalcitol.

In some embodiments, pharmaceutically active agent can includecalcimimetics, vitamins and their analogs, antibiotics, lanthanumcarbonate, lipid-lowering agents, such as a statin (e.g. LIPITOR®),other modulators of lipid profile (e.g., HDL-raising drugs),anti-hypertensives, anti-inflammatory agents (steroidal andnon-steroidal), inhibitors of pro-inflammatory cytokine (ENBRELOR®,KINERET®), and cardiovascular agents.

In some embodiments, pharmaceutically active agent includes those agentsknown in the art for treatment of inflammation orinflammation-associated disorders.

In some embodiments, pharmaceutically active agent can by abisphosphonate (Alendronate, Risendronate, Ibandronate, Zoledronicacid).

In some embodiments, pharmaceutically active agent can by ahormone-related agent.

In some embodiments, the pharmaceutically active agent is ananti-inflammatory agent. Exemplary anti-inflammatory agents include, butare not limited to, non-steroidal anti-inflammatory drugs (NSAIDs—suchas aspirin, ibuprofen, or naproxen, coricosteroids (such as presnisone),anti-malarial medication (such as hydrochloroquine), methotrexrate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamise,mycophenolate, and inhibitors of pro-inflammatory signaling pathways.

In some embodiments, the pharmaceutically active agent is an immuneresponse modulator. As used herein, the term “immune response modulator”refers to compound (e.g., a small-molecule, antibody, peptide, nucleicacid, or gene therapy reagent) that inhibits autoimmune response in asubject. Without wishing to be bound by theory, an immune responsemodulator inhibits the autoimmune response by inhibiting the activity,activation, or expression of inflammatory cytokines (e.g., IL-12, IL-23or IL-27), or STAT-4. Exemplary immune response modulators include, butare not limited to, members of the group consisting of Lisofylline (LSF)and the LSF analogs and derivatives described in U.S. Pat. No.6,774,130, contents of which are herein incorporated by reference intheir entirety.

In some embodiments, the pharmaceutically active agent is an antibioticagent. The term “antibiotic” is used herein to describe a compound orcomposition which decreases the viability of a microorganism, or whichinhibits the growth or reproduction of a microorganism. As used in thisdisclosure, an antibiotic is further intended to include anantimicrobial, bacteriostatic, or bactericidal agent. Exemplaryantibiotics include, but are not limited to, penicillin, cephalosporins,penems, carbapenems, monobactams, aminoglycosides, sulfonamides,macrolides, tetracyclins, lincosides, quinolones, chloramphenicol,vancomycin, metronidazole, rifampin, isoniazid, spectinomycin,trimethoprim, sulfamethoxazole, and the like.

The dosage regimen for treating a disease condition with the combinationtherapy disclosed herein can be selected in accordance with a variety offactors, including the type, age, weight, sex and medical condition ofthe patient, the severity of the disease, the route of administration,and the particular compound employed, and thus can vary widely.

In some embodiments, the inhibitor can be administered in conjunctionwith surgical and non-surgical treatments. In some embodiments, themethods disclosed herein can be practiced in injunction with dialysis.

Vascular calcification, a well-recognized and common complication of CRD(also known as chronic kidney disease (CKD)), increases the risk ofcardiovascular morbidity and mortality (Giachelli, C. J. Am. Soc.Nephrol. 15: 2959-64, 2004; Raggi, P. et al. J. Am. Coll. Cardiol. 39:695-701, 2002). While the causes of vascular calcification in CKD remainto be elucidated, associated risk factors include age, gender, calciumand phosphate imbalance, increased serum phosphate levels, hypertension,time on dialysis, diabetes and glucose intolerance, obesity,dyslipidemia, and cigarette smoking (Zoccali C. Nephrol. Dial.Transplant 15: 454-7, 2000). These conventional risk factors, however,do not adequately explain the high mortality rates from cardiovascularcauses in the patient population. Recent observations suggest thatcertain abnormalities in calcium and phosphorus metabolism, resulting ina raised serum calcium-phosphorus product contribute to the developmentof arterial/valvular calcification, and possibly to cardiovasculardisease, in patients with end-stage renal disease (Goodman, W. et al. N.Engl. J. Med. 342: 1478-83, 2000; Guérin, A. et al. Nephrol. Dial.Transplant 15: 1014-21, 2000; Vattikuti, R. & Towler, D. Am. J. Physiol.Endocrinol. Metab. 286: E686-96, 2004). Clinical reports further suggestthat elevated serum phosphate concentrations are associated with asubstantially greater risk of end-stage CKD, and that risk increases upto 5-fold for each 1.0-mg/dL increment in the mean serum phosphateconcentration (Mazhar, A R et al. Kidney Int 60: 324-332, 2001).

Another hallmark of advanced CKD is secondary hyperparathyroidism (HPT),characterized by elevated parathyroid hormone (PTH) levels anddisordered mineral metabolism. The elevations in calcium, phosphorus,and Ca.times.P observed in patients with secondary HPT have beenassociated with an increased risk of vascular calcification (Chertow, G.et al. Kidney Int. 62: 245-52, 2002; Goodman, W. et al. N. Engl. J. Med.342: 1478-83, 2000; Raggi, P. et al. J. Am. Coll. Cardiol. 39: 695-701,2002). Commonly used therapeutic interventions for secondary HPT, suchas calcium-based phosphate binders and doses of active vitamin D sterolscan result in hypercalcemia and hyperphosphatemia (Chertow, G. et al.Kidney Int. 62: 245-52, 2002; Tan, A. et al. Kidney Int 51: 317-23,1997; Gallieni, M. et al. Kidney Int 42: 1191-8, 1992), which areassociated with the development or exacerbation of cardiovascularcalcification.

Vascular calcification is an important and potentially seriouscomplication of chronic renal failure. Two distinct patterns of vascularcalcification have been identified (Proudfoot, D & Shanahan, C. Herz 26:245-51, 2001), and it is common for both types to be present in uremicpatients (Chen, N. & Moe, S. Semin Nephrol 24: 61-8, 2004). The medialcalcification, occurs in the media of the vessel in conjunction with aphenotypic transition of SMCs into osteoblast-like cells, whileatherogenesis, is associated with lipid-laden macrophages and intimalhyperplasia.

Medial wall calcification can develop in relatively young persons withchronic renal failure, and it is common in patients with diabetesmellitus even in the absence of renal disease. The presence of calciumin the medial wall of arteries distinguishes this type of vascularcalcification from that associated with atherosclerosis (Schinke T. &Karsenty G. Nephrol Dial Transplant 15: 1272-4, 2000). Atheroscleroticvascular calcification occurs in atheromatous plaques along the intimallayer of arteries (Farzaneh-Far A. JAMA 284:1515-6, 2000). Calcificationis usually greatest in large, well developed lesions, and it increaseswith age (Wexler L. et al. Circulation 94: 1175-92, 1996; Rumberger J.et al. Mayo Clin Proc 1999; 74: 243-52.). The extent of arterialcalcification in patients with atherosclerosis generally corresponds toseverity of disease. Unlike medial wall calcification, atheroscleroticvascular lesions, whether or not they contain calcium, impinge upon thearterial lumen and compromise blood flow. The localized deposition ofcalcium within atherosclerotic plaques may happen because ofinflammation due to matrix vesicles release, apoptosis or oxidizedlipids and other oxidative stresses and infiltration by monocytes andmacrophages (Berliner J. et al. Circulation 91: 2488-96, 1995).

Some patients with end-stage renal disease develop a severe form ofocclusive arterial disease called calciphylaxis or calcific uremicarteriolopathy. This syndrome is characterized by extensive calciumdeposition in small arteries (Gipstein R. et al. Arch Intern Med 136:1273-80, 1976; Richens G. et al. J Am Acad. Dermatol. 6: 537-9, 1982).In patients with this disease, arterial calcification and vascularocclusion lead to tissue ischemia and necrosis. Involvement ofperipheral vessels can cause ulceration of the skin of the lower legs organgrene of the digits of the feet or hands. Ischemia and necrosis ofthe skin and subcutaneous adipose tissue of the abdominal wall, thighsand/or buttocks are features of a proximal form of calcific uremicarteriolopathy (Budisavljevic M. et al. J Am Soc Nephrol. 7: 978-82,1996; Ruggian J. et al. Am. J. Kidney Dis. 28: 409-14, 1996). Thissyndrome occurs more frequently in obese individuals, and women areaffected more often than men for reasons that remain unclear (Goodman W.J. Nephrol. 15(6): S82-S85, 2002).

As used herein, the term “cardiovascular calcification” means formation,growth or deposition of extracellular matrix hydroxyapatite (calciumphosphate) crystal deposits in blood vessels. Cardiovascularcalcification encompasses coronary, aortic, arterial, vein graft,tissue-engineered vessel, and other blood vessel calcification as wellas aortic valve and mitral annulus calcification. The term includesatherosclerotic and medial wall calcification in vessels.

As used herein, the term “atherosclerotic calcification” means vascularcalcification occurring in atheromatous plaques along the intimal layerof arteries.

Intimal calcification occurs within the perimeter of the internalelastic lamina as part of the atherosclerotic plaque and is often seenas discrete, punctate lesions on radiographs. It is associated withinflammatory cells, lipid, and vascular smooth muscle cells.

As used herein, the terms “medial calcification,” “medial wallcalcification,” and “Monckeberg's sclerosis,” mean calcificationcharacterized by the presence of calcium in the medial wall of arteries.

Methods of detecting and measuring cardiovascular calcification are wellknown in the art. In one aspect, methods of measuring calcificationinclude direct methods of detecting and measuring extent ofcalcium-phosphorus depositions in blood vessels.

In one aspect, direct methods of measuring cardiovascular calcificationcomprise in vivo imaging methods such as plain film roentgenography,coronary arteriography; fluoroscopy, including digital subtractionfluoroscopy; cinefluorography; conventional, helical, and electron beamcomputed tomography; intravascular ultrasound (IVUS); magnetic resonanceimaging; and transthoracic and transesophageal echocardiography.Fluoroscopy and EBCT are most commonly used to detect calcificationnoninvasively, while cinefluorography and IVUS are used by coronaryinterventionalists to evaluate calcification in specific lesions beforeangioplasty. Transthoracic echocardiography is commonly used to detectaortic valve calcification.

In one aspect, cardiovascular calcification can be detected by plainfilm roentgenography. The advantage of this method is availability ofthe film and the low cost of the method, however, the disadvantage isits low sensitivity. Kelley M. & Newell J. Cardiol Clin. 1: 575-595,1983.

In another aspect, fluoroscopy can be used to detect calcification incoronary arteries. Although fluoroscopy can detect moderate to largecalcifications, its ability to identify small calcific deposits is low(Loecker et al. J Am Coll Cardiol. 19: 1167-1172, 1992). Fluoroscopy iswidely available in both inpatient and outpatient settings and isrelatively inexpensive, but it has several disadvantages. In addition toonly a low to moderate sensitivity, fluoroscopic detection of calcium isdependent on the skill and experience of the operator as well as thenumber of views studied. Other important factors include variability offluoroscopic equipment, the patient's body habitus, overlying anatomicstructures, and overlying calcifications in structures such as vertebraeand valve annuli. With fluoroscopy, quantification of calcium is notpossible, and film documentation is not commonly obtained.

In yet another aspect, cardiovascular calcification can be detected byconventional computed tomography (CT). Because calcium attenuates thex-ray beam, computed tomography (CT) is extremely sensitive in detectingcardiovascular calcification. While conventional CT appears to havebetter capability than fluoroscopy to detect coronary arterycalcification, its limitations are slow scan times resulting in motionartifacts, volume averaging, breathing misregistration, and inability toquantify amount of plaque (Wexler et al. Circulation 94: 1175-1192,1996). Aortic valve calcification is often detected by conventional CT,particularly in the elderly (Liu et al., American Journal ofRoentgenology 186:342-349, 2006).

In a further aspect, calcification can be detected by helical or spiralcomputer tomography, which has considerably faster scan times thanconventional CT. Overlapping sections also improve calcium detection.Shemesh et al. reported coronary calcium imaging by helical CT as havinga sensitivity of 91% and a specificity of 52% when compared withangiographically significant coronary obstructive disease (Shemesh etal. Radiology 197: 779-783, 1995). However, other preliminary data haveshown that even at these accelerated scan times, and especially withsingle helical CT, calcific deposits are blurred due to cardiac motion,and small calcifications may not be seen (Baskin et al. Circulation92(suppl I): 1-651, 1995). Thus, helical CT remains superior tofluoroscopy and conventional CT in detecting calcification. Double-helixCT scanners appear to be more sensitive than single-helix scanners indetection of coronary calcification because of their higher resolutionand thinner slice capabilities (Wexler et al. Circulation 94: 1175-1192,1996)

In another aspect, Electron Beam Computed Tomography (EBCT) can be usedfor detection of cardiovascular calcification. EBCT uses an electron gunand a stationary tungsten “target” rather than a standard x-ray tube togenerate x-rays, permitting very rapid scanning times. Originallyreferred to as cine or ultrafast CT, the term EBCT is now used todistinguish it from standard CT scans because modern spiral scanners arealso achieving subsecond scanning times. For purposes of detectingcoronary calcium, EBCT images are obtained in 100 ms with a scan slicethickness of 3 mm. Thirty to 40 adjacent axial scans are obtained bytable incrementation. The scans, which are usually acquired during oneor two separate breath-holding sequences, are triggered by theelectrocardiographic signal at 80% of the RR interval, near the end ofdiastole and before atrial contraction, to minimize the effect ofcardiac motion. The rapid image acquisition time virtually eliminatesmotion artifact related to cardiac contraction. The unopacified coronaryarteries are easily identified by EBCT because the lower CT density ofperiarterial fat produces marked contrast to blood in the coronaryarteries, while the mural calcium is evident because of its high CTdensity relative to blood. Additionally, the scanner software allowsquantification of calcium area and density. An arbitrary scoring systemhas been devised based on the x-ray attenuation coefficient, or CTnumber measured in Hounsfield units, and the area of calcified deposits(Agatston et al. J Am Coll Cardiol. 15:827832, 1990). A screening studyfor coronary calcium can be completed within 10 or 15 minutes, requiringonly a few seconds of scanning time. Electron beam CT scanners are moreexpensive than conventional or spiral CT scanners and are available inrelatively fewer sites.

In one aspect, intravascular ultrasound (IVUS) can be used for detectingvascular calcification, in particular, coronary atherosclerosis (Walleret al. Circulation 85: 23052310, 1992). By using transducers withrotating reflectors mounted on the tips of catheters, it is possible toobtain cross-sectional images of the coronary arteries during cardiaccatheterization. The sonograms provide information not only about thelumen of the artery but also about the thickness and tissuecharacteristics of the arterial wall. Calcification is seen as ahyperechoic area with shadowing: fibrotic noncalcified plaques are seenas hyperechoic areas without shadowing. (Honye et al. Trends CardiovascMed. 1: 305-311, 1991). The disadvantages in use of IVUS, as opposed toother imaging modalities, are that it is invasive and currentlyperformed only in conjunction with selective coronary angiography, andit visualizes only a limited portion of the coronary tree. Althoughinvasive, the technique is clinically important because it can showatherosclerotic involvement in patients with normal findings on coronaryarteriograms and helps define the morphological characteristics ofstenotic lesions before balloon angioplasty and selection of atherectomydevices (Tuzcu et al. J Am Coll Cardiol. 27: 832-838, 1996).

In another aspect, cardiovascular calcification can be measured bymagnetic resonance imaging (MRI). However, the ability of MRI to detectcoronary calcification is somewhat limited. Because microcalcificationsdo not substantially alter the signal intensity of voxels that contain alarge amount of soft tissue, the net contrast in such calciumcollections is low. Therefore, MRI detection of small quantities ofcalcification is difficult, and there are no reports or expected rolesfor MRI in detection of early coronary artery calcification ormicrocalcification (Wexler et al. Circulation 94: 1175-1192, 1996).

In another aspect, cardiovascular calcification can be measured bytransthoracic (surface) echocardiography, which is particularlysensitive to detection of mitral and aortic valvular calcification;however, visualization of the coronary arteries has been documented onlyon rare occasions because of the limited available external acousticwindows. Transesophageal echocardiography is a widely availablemethodology that often can visualize the proximal coronary arteries (Kohet al. Int J Cardiol. 43: 202-206, 1994. Fernandes et al. Circulation88: 2532-2540, 1993).

In another aspect, cardiovascular calcification can be assessed usingnear-infrared molecular imaging with a sensitive calcium bindingmolecular imaging agent using intravital fluorescence microscope orfluorescence reflectance imaging system (Aikawa E et al 116: 2841-2850,Circulation, 2007; Aikawa E et al, 119: 1785-1794; Circulation, 2009;New E P et al. 108; 1381-1391, Circ Res, 2011). However, while molecularimaging provides high-resolution images of microcalcification inarteries and valves, the system has been used only in animal studies.

In another aspect, cardiovascular calcification can be assessed inhumans using 18F-Sodium Fluoride (18F—NaF) Positron Emission Tomography(PET) (George et al., J Am Coll Cardiol. 59:1549-50, 2012; Dweck et al.,J Am Coll Cardiol. 59:1539-1548, 2012; Aikawa E, et al., Circulation125:9-11, 2012; Dweck et al., Circulation 125:76-86, 2012).

In another aspect, vascular calcification can be assessed ex vivo by VonKossa method. This method relies upon the principle that silver ions canbe displaced from solution by carbonate or phosphate ions due to theirrespective positions in the electrochemical series. The argentaffinreaction is photochemical in nature and the activation energy issupplied from strong visible or ultra-violet light. Since thedemonstrable forms of tissue carbonate or phosphate ions are invariablyassociated with calcium ions the method may be considered asdemonstrating sites of tissue calcium deposition.

Other methods of direct measuring calcification may include, but are notlimited to, immunofluorescent staining and densitometry. In anotheraspect, methods of assessing vascular calcification include methods ofmeasuring determinants and/or risk factors of vascular calcification.Such factors include, but are not limited to, serum levels ofphosphorus, calcium, and Ca×P product, parathyroid hormone (PTH),low-density lipoprotein cholesterol (LDL), high-density lipoproteincholesterol (HDL), triglycerides, and creatinine. Methods of measuringthese factors are well known in the art. Other methods of assessingvascular calcification include assessing factors of bone formation. Suchfactors include bone formation markers such as bone-specific alkalinephosphatase (BSAP), osteocalcin (OC), osteopontin (OPN), osteonectin(ON), sclerostin (SOST), dickkopf-1 (DKK1) carboxyterminal propeptide oftype I collagen (PICP), and amino terminal propeptide of type I collagen(PINP); serum bone resorption markers such as cross-linked C-telopeptideof type I collagen (ICTP), tartrate-resistant acid phosphatase, TRACPand TRAP5B, N-telopeptide of collagen cross-links (NTx), andC-telopeptide of collagen cross-links (CTx); osteogenic transcriptionmarkers such as RUNX2/Cbfal, Osterix, Msx-2, and urine bone resorptionmarkers, such as hydroxyproline, free and total pyridinolines (Pyd),free and total deoxypyridinolines (Dpd), N-telopeptide of collagencross-links (NTx), and C-telopeptide of collagen cross-links (CTx).

The work reported herein shows that exogenous sortilin 1 increasesmatrix mineralization and serum sortilin 1 levels are elevated inatherosclerotic mice. Accordingly, blood levels of sortilin 1 can beused a biomarker for severity of calcification or CRD. Thus, in oneaspect provided herein is an assay for determining level or severity ofcalcification or CRD in a subject. The assay comprises: (a) subjecting atest sample of a subject to at least one analysis to determine the levelof sortilin 1, wherein an increased level of sortilin 1 relative to areference or control sample indicates that the subject has elevatedcalcification levels, at risk of developing calcification, has CRD, oris at risk of developing CRD. The level of sortilin 1 can be determinedfrom the amount of sortilin1 itself or a nucleic acid encoding sortilin1.

The elevated or increased level of sortilin 1 can be at least 10% higherthan a reference or control level. The Reference or control level can besortilin 1 level in a healthy subject or a level determined previouslyfrom the same subject. For the avoidance of any doubt, the terms“increased” and “elevated” with reference to sortilin 1 level means anincrease of at least 10% as compared to a reference level, for examplean increase of at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% increase or any increase between 10-100% as compared toa reference level, or at least about a 2-fold, or at least about a3-fold, or at least about a 4-fold, or at least about a 5-fold or atleast about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level or control level.

Collections of test samples for at least one analysis performed in theassays and/or methods described herein are well known to those skilledin the art. In some embodiments, a test sample subjected to analysisperformed in the assays and/or methods described herein are derived froma biological sample of a subject. The term “biological sample” as usedherein denotes a sample taken or isolated from a biological organism,e.g., cell lysate, a homogenate of a tissue sample from a subject or afluid sample from a subject. The term “biological sample” also includesuntreated or pre-treated (or pre-processed) biological samples. In someembodiments, the biological sample can be a biological fluid, including,but not limited to, blood (including whole blood, plasma, cord blood andserum), lactation products (e.g., milk), amniotic fluids, sputum,saliva, urine, semen, cerebrospinal fluid, bronchial aspirate,perspiration, mucus, liquefied feces, synovial fluid, lymphatic fluid,tears, tracheal aspirate, and fractions thereof. In other embodiments,the biological sample can include cell lysate and fractions thereof. Forexample, cells (such as red blood cells, platelets, white blood cellsand any cells circulating in the biological fluid described herein) canbe harvested and lysed to obtain a cell lysate. In some embodiments, thebiological sample is blood, plasma, or serum.

A “biological sample” can contain cells from subject, but the term canalso refer to non-cellular biological material, such as non-cellularfractions of blood, saliva, or urine, that can be used to measuresortilin 1 levels. In some embodiments, the sample is from a resection,biopsy, or core needle biopsy. In addition, fine needle aspirate samplescan be used. Samples can be either paraffin-embedded or frozen tissue.

The biological sample can be a clinical sample. A “clinical sample” is asample derived from a human subject. A biological sample can also bereferred to as a “subject sample.” A test biological sample is thebiological sample that has been the object of analysis, monitoring, orobservation. A control biological sample can be either a positive or anegative control for the test biological sample. Often, the controlbiological sample contains the same types of tissues, cells andbiological fluids as that of the test biological sample. The sample canbe obtained by removing a sample of cells from a subject, but can alsobe accomplished by using previously isolated cells (e.g. isolated byanother person). In addition, the biological sample can be freshlycollected or a previously collected sample.

In some embodiments, the test sample or the biological sample can be afrozen biological sample, e.g., a frozen tissue or fluid sample such asurine, blood, serum or plasma. The frozen sample can be thawed beforeemploying methods, assays and systems of the invention. After thawing, afrozen sample can be centrifuged before being subjected to methods,assays and systems of the invention.

In some embodiments, a test sample or a biological sample can be anucleic acid product amplified after polymerase chain reaction (PCR).The nucleic acid product of the instant invention include DNA, RNA andmRNA and can be isolated from a particular biological sample using anyof a number of procedures, which are well-known in the art, theparticular isolation procedure chosen being appropriate for theparticular biological sample. Methods of isolating and analyzing nucleicacid variants as described above are well known to one skilled in theart and can be found, for example in the Molecular Cloning: A LaboratoryManual, 3rd Ed., Sambrook and Russel, Cold Spring Harbor LaboratoryPress, 2001.

In some embodiments, the test sample or the biological sample can betreated with a chemical and/or biological reagent. Chemical and/orbiological reagents can be employed to protect and/or maintain thestability of the sample, including biomolecules (e.g., nucleic acid andprotein) therein, during processing. One exemplary reagent is a proteaseinhibitor, which is generally used to protect or maintain the stabilityof protein during processing. In addition, or alternatively, chemicaland/or biological reagents can be employed to release nucleic acid orprotein from the sample.

The skilled artisan is well aware of methods and processes appropriatefor pre-processing of test or biological samples, e.g., blood, requiredfor determination of sortilin 1 levels as described herein.

In some embodiments, the test sample or biological sample is a bloodsample, e.g., whole blood, plasma, and serum. In some embodiments, thetest sample or biological sample is a whole blood sample. In someembodiments, the test sample or biological sample is a serum sample. Insome embodiments, the test sample or biological sample is a plasmasample. In some embodiments, the blood sample can be allowed to dry atroom temperature from about 1 hour to overnight, or in the refrigerator(low humidity) for up to several months before subjected to analysis

To collect a blood sample, by way of example only, the patient's bloodcan be drawn by trained medical personnel directly into anti-coagulantssuch as citrate, EDTA PGE, and theophylline. The whole blood can beseparated into the plasma portion, the cells, and platelets portion byrefrigerated centrifugation at 3500 g for 2 minutes. Aftercentrifugation, the supernatant is the plasma and the pellet is RBC.Since platelets have a tendency to adhere to glass, it is preferred thatthe collection tube be siliconized. Another method of isolating redblood cells (RBCs) is described in Best, C A et al., 2003, J. LipidResearch, 44:612-620.

Alternatively, serum can be collected from the whole blood. By way ofexample, about 15 mL of whole blood can be drawn for about 6 mL ofserum. The blood can be collected in a hard plastic or glass tube; bloodwill not clot in soft plastic. The whole blood is allowed to stand atroom temperature for 30 minutes to 2 hours until a clot has formed.Then, clot can be carefully separated from the sides of the containerusing a glass rod or wooden applicator stick and the rest of the samplecan be left overnight at 4° C. After which, the sample can becentrifuged, and the serum can be transferred into a clean tube. Theserum can be clarified by centrifugation at 1000 g for 10 minutes at 4°C. The serum can be stored at −80° C. before analysis. In suchembodiments, carotenoids may not be stable for long periods of time.Detailed described of obtaining serum using collection tubes can befound in U.S. Pat. No. 3,837,376 and is incorporated by reference. Bloodcollection tubes can also be purchased from BD Diagnostic Systems,Greiner Bio-One, and Kendall Company.

The whole blood can be first separated into platelet-rich plasma andcells (white and red blood cells). Platelet rich plasma (PRP) can beisolated from the blood centrifugation of citrated whole blood at 200 gfor 20 minutes. The platelet rich plasma is then transferred to a freshpolyethylene tube. This PRP is then centrifuged at 800 g to pellet theplatelets and the supernatant (platelet poor plasma [PPP]) can be savedfor analysis, e.g., by ELISA, at a later stage. Platelets can be thengently re-suspended in a buffer such as Tyrodes buffer containing 1 U/mlPGE2 and pelleted by centrifugation again. The wash can be repeatedtwice in this manner before removing the membrane fraction of plateletsby centrifugation with Triton X, and lysing the pellet of platelet forplatelet-derived PF4 analyses. Platelets can be lysed using 50 mM TrisHCL, 100-120 mM NaCl, 5 mM EDTA, 1% Igepal and Protease Inhibitor Tablet(complete TM mixture, Boehringer Manheim, Indianopolis, Ind.).

Methods of measuring the amount or presence of specific proteins andnucleic acids levels in biological samples are well known to one ofskill in the art. For example, if the analyte is a protein, i.e.,sortilin 1, the test sample can be subjected to at least one analysisselected from the group consisting of western blot, enzyme linkedabsorbance assay, mass spectrometry, immunoassay, flow cytometry,immunohistochemical analysis, and any combinations thereof. For example,a western blot, ELISA, immunoassay, flow cytometry, orimmunohistochemical analysis can be performed using an anti-Sortilin 1antibody. Exemplary anti-Sortilin 1 antibodies for using in the assaydisclosed herein include, but are not limited to, NTR3 (E-9), NTR3(G-11), NTR3 (C-19), NTR3 (F-15), NTR (H-300), and NTR3 (C-20), NTR3(N-17) available from Santa Cruz Biotechnology; SORT1 purified MAxPabmouse polyclonal antibody, SORT 1 polyclonal antibody, SORT lmonoclonalantibody (M01, clone 1B3), and SORT 1 polyclonal antibody (A01)available from Abnova; Neurotensin Receptor 3 (NTR3, Sortilin, 100 kD NTReceptor, Glycoprotein 95, Gp95, NT3, OTTHUMP00000013784, Sortilin 1,Sortilin-1, SORT1) available from US Biological; Anti-Sortilin availablefrom EMD Millipore; Human/Mouse Soritlin Mab (Clone 334708); HumanSortilin Affinity Purified Polyclonal Ab; and Human Sortilin AffinityPurified Polyclonal Ab available from R and Systems; Sortilin antibodyavailable from Biorbyst; Sortilin 1 antibody [C1C3] available fromGeneTex; Anti-SORT1/Sortilin available from LifeSpan BioSciences; SORT 1available from Proteintech; Sortilin 1, Sortilin, Neurotensin Receptor 3available from Thermo Scientific Pierce Products; monoclonal anti-SORT1antibody produced in mouse and anti-SORT1 antibody produced in rabbitavailable from Sigma-Aldrich.

If the analyte is a nucleic acid, e.g., a nucleic acid encoding sortilin1, the test sample can be subjected to at least one analysis selectedfrom the group consisting of probe hybridization, primer extension,amplification, sequencing, 5′ nuclease digestion, molecular beaconassay, DNA chip analysis, oligonucleotide ligation assay, size analysis,single-stranded conformation polymorphism, polymerase chain reaction(PCR), real-time quantitative PCR, and any combinations thereof. In oneembodiment, PCD analysis can be performed using Hs00361747_ml TaqManprobes from Life Technologies. In some embodiments, the PCR analysis canbe performed with a PCR primer pair having a first primer with anucleotide sequence comprising at least 18 consecutive nucleotides ofhuman SORT 1 (NCBI Reference Sequence: NM_002959.4 or NM_002959.5) and asecond primer with a nucleotide sequence comprising a sequencecomplementary to at least at least 18 consecutive nucleotides of humanSORT 1 (NCBI Reference Sequence: NM_002959.4 or NM_002959.5).

In some embodiments, the assay further comprises administering a therapyor an agent (e.g., a therapeutic agent) for reducing or inhibitingvascular calcification or treating a vascular calcification-relatedcondition to the subject having an increased level of Sortilin 1 in theassay described herein. Agents and therapies useful for reducing orinhibiting calcification or treating vascular calcification-relatedconditions include, but are not limited to, statins, bisphosphonates,phosphate binders, mineralocorticoid receptor antagonists, and anycombinations thereof. Some exemplary compositions and methods forreducing or inhibiting vascular calcification or treating vascularcalcification-related conditions are described in PCT App. Publ. No.WO2000003677, No. WO2000033865, No. WO2001003774, No. WO2001070320, No.WO2002009683, No. WO2003013420, No. WO2004019923, No. WO2005044189, No.WO2006102061, No. WO2006122046, No. WO2007047969, No. WO2007112280, No.WO2008060139, No. WO2008115469, No. WO2008116215, No. WO2009017863, No.WO2009072132, No. WO2009102966, No. WO2010083613, No. WO2011005841, No.WO2011123518, No. WO2011133855, No. WO2012065059, No. WO2012100229 andNo. WO2013006372; and U.S. Pat. No. 7,422,607, contents of all of whichare incorporated herein by reference in their entireties. Anti-RANKLantibody, Anti-sclerostin antibody and anti-Wnt antibody are used forthe treatment of the bone in preclinical studies. These antibodies couldalso be used in combination with sortilin 1 therapy.

Also provided herein is a composition or combination comprising anisolated sample obtained from a subject and a first reagent to reactwith sortilin 1. The sample can be a sample suspected of comprising anelevated level of sortilin 1. In some embodiments, the combinationfurther comprises a second reagent that produces a signal in thepresence of sortilin1 and the first reagent.

The first and second reagent can be selected independently from thegroup consisting of small organic or inorganic molecules; saccharines;oligosaccharides; polysaccharides; peptides; proteins; peptide analogsand derivatives; peptidomimetics; glycoproteins, glycopeptides;antibodies and antigen binding fragments thereof; nucleic acids; nucleicacid analogs and derivatives; lipids, an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal cells; animaltissues; naturally occurring or synthetic compositions; and anycombinations thereof. In some embodiments, the first reagent is asubstrate that sortilin 1 acts on, a molecule that binds with sortilin1, a molecule that inhibits binding of sortilin to a second molecule;and any combinations thereof.

In some embodiments, the first or the second reagent can comprise alabel to produce a signal if sortilin 1 is present in the sample. Amountor level of the signal produced in the presence of sortilin can becorrelated to amount or level of sortilin in the sample. Exemplarylabels include, but are not limited to, radiolabels, chromophores,fluorophores, chemiluminescent precursors, chemiluminescent reactants,and the like.

The disclosure also provides a system comprising thecomposition/combination discussed above.

Computer systems for use in any aspects of the assay for determiningsortilin 1 levels described herein are also provided. For example, oneembodiment provided herein is a computer system for obtaining data fromat least one test sample obtained from at least one subject. The systemcomprises: (a) a determination module configured to receive at least onetest sample from the subject and perform at least one analysis on atleast one test sample to determine the level or amount of sortilin 1 ora nucleic acid encoding sortiline 1; (b) a storage device configured tostore data output from the determination module; and (c) a displaymodule for displaying a content based in part on the data output fromthe determination module, wherein the content comprises a signalindicative of the amount or level of sortilin 1, and optionally thepresence or absence of calcification or CRD.

In some embodiments, the determination module can further comprise acomparison module adapted to compare the data output from thedetermination module with reference data stored on the storage device.

In some embodiments, the storage device can be further configured tostore information of at least one subject, for example, previouslydetermined sortilin 1 level(s) of at least one subject.

In some embodiments, the content displayed on the display module canfurther comprises a signal indicative of whether the sortilin 1 level isat least 10% higher than a reference of control level.

In some embodiments, the content displayed on the display module canfurther comprise a signal indicative of the subject recommended toreceive a treatment regimen for inhibiting calcification.

A computer readable medium having computer readable instructionsrecorded thereon to define software modules for implementing a method ona computer is also provided herein. In one embodiment, the computerreadable storage medium comprises: (a) instructions for comparing thedata stored on a storage device with reference data to provide acomparison result, wherein the comparison identifies elevated levels ofsortilin 1; and (b) instructions for displaying a content based in parton the data output from the determination module, wherein the contentcomprises a signal indicative of elevated level of sortilin 1, andoptionally the presence or absence of calcification or CRD.

In some embodiments, the computer readable medium can further compriseinstructions to determine or calculate if the subject has a sortilin 1level that is at least 10% higher than a reference or control level.

Exemplary embodiments of the invention can be described by any one ofthe following numbered paragraphs:

-   1. A method for inhibiting calcification of a smooth muscle cell    (SMC), the method comprising contacting a compound with a SMC,    wherein the compound inhibits: (i) activity or amount of sortilin 1    in a vascular smooth muscle cell or valvular interstitial    cells; (ii) expression of a nucleic acid encoding sortilin 1 in a    smooth muscle cell; or (iii) phosphorylation of sortilin 1.-   2. The method of paragraph 1, the compound is selected from the    group consisting of small organic or inorganic molecules;    saccharines; oligosaccharides; polysaccharides; peptides; proteins;    peptide analogs and derivatives; peptidomimetics; glycoproteins,    glycopeptides; antibodies and antigen binding fragments thereof;    nucleic acids; nucleic acid analogs and derivatives; an extract made    from biological materials such as bacteria, plants, fungi, or animal    cells; animal tissues; naturally occurring or synthetic    compositions; and any combinations thereof.-   3. The method of any of paragraphs 1-2, wherein the compound is a    siRNA or an antibody.-   4. The method of any of paragraphs 1-3, wherein the compound    inhibits the expression of the nucleic acid encoding sortilin 1 by    at least 10% relative to a control or reference level.-   5. The method of any of paragraphs 1-4, wherein the compound    inhibits the activity of sortilin 1 by at least 10% relative to a    control or reference level.-   6. The method of any of paragraphs 1-5, wherein the compound    decreases the amount of sortilin 1 in the SMC by at least 10%    relative to a control or reference level.-   7. The method of any of paragraphs 1-6, wherein the compounds    reduces tissue non-specific alkaline phosphatase activity (TNAP) by    at least 10% relative to a control or reference level.-   8. The method of any of paragraphs 1-7, wherein the compound    increases phosphate regulating endopeptidase (PHEX) expression by at    least 10% relative to a control or reference level.-   9. The method of any of paragraphs 1-8, wherein the compound    decreases matrix mineralization by at least 10% relative to a    control or reference level.-   10. The method of any of paragraphs 1-9, wherein the compound    increases expression level of microRNA 125b by at least 10% relative    to a control or reference level.-   11. The method of any of paragraphs 1-10, wherein the compound    decreases the association of sortilin 1 to non-specific alkaline    phosphatase activity (TNAP), Caveolin-1.-   12. The method of any of paragraphs 1-11, wherein the nucleic acid    encoding sortilin 1 is mRNA.-   13. The method of any of paragraphs 1-12, wherein said contacting is    in vitro.-   14. The method of any of paragraphs 1-13, wherein said contacting is    in vivo.-   15. The method of paragraph 14, wherein said contacting is in a    mammal.-   16. The method of paragraph 14 or 15, wherein said contacting is in    a human.-   17. The method of any of paragraphs 14-16, wherein said contacting    is in a subject in need of inhibition of calcification.-   18. The method of paragraph 17, wherein said calcification is    cardiovascular calcification.-   19. The method of paragraph 17 or 18, wherein said calcification is    valvular or arterial calcification.-   20. The method of any of paragraphs 17-19, wherein the severe renal    failure, or has a transcatheter aortic valve implantation, or has    chronic coronary atherosclerosis, or has aortic stenosis.-   21. The method of any of paragraphs 17-20, wherein the subject has    mineral imbalance or a calcium/phosphate disorder, including chronic    renal disease, hemodialysis and type II diabetes; arterio-venous    grafts/shunts; arterial and vein grafts; tissue engineered vascular    and valvular implants; Paget's disease, rheumatoid arthritis,    osteoporosis or osteoarthritis.-   22. A method for inhibiting calcification or a clinical complication    arising therefrom in a subject, the method comprising administering    a therapeutically effective amount of a compound to a subject in    need thereof, wherein the compound inhibits: (i) activity,    phosphorylation, or amount of sortilin 1 in a SMC; (ii) expression    of a nucleic acid encoding sortilin 1 in a SMC; or (iii)    phosphorylation of sortilin 1.-   23. The method of paragraph 22, wherein the compound is selected    from the group consisting of small organic or inorganic molecules;    saccharines; oligosaccharides; polysaccharides; peptides; proteins;    peptide analogs and derivatives; peptidomimetics; glycoproteins,    glycopeptides; antibodies and antigen binding fragments thereof;    nucleic acids; nucleic acid analogs and derivatives; an extract made    from biological materials such as bacteria, plants, fungi, or animal    cells; animal tissues; naturally occurring or synthetic    compositions; and any combinations thereof.-   24. The method of any of paragraphs 22-23, wherein the compound is a    siRNA or an antibody.-   25. The method of any of paragraphs 22-24, wherein the compound    inhibits the expression of the nucleic acid encoding sortilin 1 by    at least 10% relative to a control or reference level.-   26. The method of any of paragraphs 22-25, wherein the compound    inhibits the activity of sortilin 1 by at least 10% relative to a    control or reference level.-   27. The method of any of paragraphs 22-26, wherein the compound    decreases the amount of sortilin 1 in the SMC by at least 10%    relative to a control or reference level.-   28. The method of any of paragraphs 22-27, wherein the compounds    reduces tissue non-specific alkaline phosphatase activity by at    least 10% relative to a control or reference level.-   29. The method of any of paragraphs 22-28, wherein the compound    increases phosphate regulating endopeptidase expression by at least    10% relative to a control or reference level.-   30. The method of any of paragraphs 22-29, wherein the compound    decreases matrix mineralization by at least 10% relative to a    control or reference level.-   31. The method of any of paragraphs 22-30, wherein the compound    increases expression level of microRNA 125b by at least 10% relative    to a control or reference level.-   32. The method of any of paragraphs 22-31, wherein the compound    decreases the association of sortilin 1 to non-specific alkaline    phosphatase activity (TNAP), Caveolin-1.-   33. The method of any of paragraphs 22-32 wherein the nucleic acid    encoding sortilin 1 a mRNA.-   34. The method of any of paragraphs 22-33, wherein said    administering is implant, injection, infusion, instillation,    implantation, or ingestion-   35. The method of any of paragraphs 22-34, wherein the    therapeutically effective amount is from about 1 μg/kg to about 150    mg/kg of body weight.-   36. The method of any of paragraphs 22-35, wherein said    administering is once a day.-   37. The method of any of paragraphs 22-36, wherein the subject is a    mammal.-   38. The method of any of paragraphs 22-37, wherein said    calcification is cardiovascular calcification.-   39. The method of any of paragraphs 22-38, wherein said    calcification is valvular or arterial calcification.-   40. The method of any of paragraphs 22-39, wherein the subject has    severe renal failure, or has a transcatheter aortic valve    implantation, or has chronic coronary atherosclerosis, or has aortic    stenosis.-   41. The method of any of paragraphs 22-41, wherein the subject has    mineral imbalance or a calcium/phosphate disorder, including chronic    renal disease, hemodialysis and type II diabetes; arterio-venous    grafts/shunts; arterial and vein grafts; tissue engineered vascular    and valvular implants; Paget's disease, rheumatoid arthritis,    osteoporosis or osteoarthritis.-   42. The method of any of paragraphs 22-41, wherein the clinical    complication is acute myocardial infraction, stroke, and the like.-   43. The method of any of paragraphs 22-42, wherein the subject has    an increased level of sortilin relative to a reference level.-   44. A method comprising:    -   subjecting a biological sample from a subject to at least one        analysis to detect level of sortilin 1 in the biological sample;    -   comparing the level of sortilin 1 in the sample to a reference        level;    -   identifying the subject as having calcification or at risk of        developing calcification if the level in the sample is higher        than the reference level; and    -   administering a therapy for inhibiting or reducing calcification        or for treating a vascular calcification-related condition such        as CRD, diabetes, aortic valve stenosis, and some genetic        disorders (e.g., Gaucher's disease).-   45. The method of paragraph 44, wherein said therapy inhibits: (i)    activity or amount of sortilin 1 in a SMC; (ii) expression of a    nucleic acid encoding sortilin 1 in a SMC; or (iii) phosphorylation    of sortilin 1.-   46. An assay for determining calcification in a subject, the assay    comprising subjecting a biological sample from a subject to at least    one analysis to detect level of sortilin 1 in the biological sample,    wherein an increased level of sortilin 1 relative to a reference    level indicates the subject has calcification, is at risk of    developing calcification, has chronic renal disease (CRD), or is at    risk of developing CRD.-   47. The assay of paragraph 46, wherein said at least one analysis is    selected from the group consisting of western blot, enzyme linked    absorbance assay, mass spectrometry, immunoassay, flow cytometry,    immunohistochemical analysis, probe hybridization, primer extension,    amplification, sequencing, 5′ nuclease digestion, molecular beacon    assay, DNA chip analysis, oligonucleotide ligation assay, size    analysis, single-stranded conformation polymorphism, polymerase    chain reaction (PCR), real-time quantitative PCR, and any    combinations thereof.-   48. The assay of paragraph 46 or 47, wherein the level of sortilin 1    is at least 10% higher than the reference or control level.-   49. The assay of any of paragraphs 46-48, wherein the biological    sample is a blood sample.-   50. In combination, an isolated sample obtained from a subject,    wherein the sample is suspected of comprising an elevated level of    sortilin 1 and a reagent to react with sortilin 1.-   51. The combination of paragraph 50, wherein the isolated sample is    a blood sample.-   52. The combination of paragraph 50 or 51, wherein the reagent is    selected from the group consisting of small organic or inorganic    molecules; saccharines; oligosaccharides; polysaccharides; peptides;    proteins; peptide analogs and derivatives; peptidomimetics;    glycoproteins, glycopeptides; antibodies and antigen binding    fragments thereof; nucleic acids; nucleic acid analogs and    derivatives; lipids, an extract made from biological materials such    as bacteria, plants, fungi, or animal cells; animal tissues;    naturally occurring or synthetic compositions; and any combinations    thereof.-   53. The combination of any of paragraphs 50-52, wherein the reagent    is a substrate that sortilin 1 acts on, a molecule that binds with    sortilin 1, a molecule that inhibits binding of sortilin to a second    molecule; and any combinations thereof-   54. The combination of any of paragraphs 50-53, wherein the reagent    further comprises a label to produce a signal so as to detect the    elevated level of the sortilin 1 in the isolated sample.-   55. The combination of paragraph 54, wherein the label is any one or    more of a radiolabel, a chromophore, a fluorophore, a    chemiluminescent precursor, a chemiluminescent reactants, or a    combination thereof.-   56. The combination of any of paragraphs 50-55, wherein the elevated    level is at least 10% higher than a control or reference level.-   57. A system comprising:    -   (a) an isolated sample obtained from a subject, wherein the        sample is suspected of comprising an elevated level of sortilin        1; and    -   (b) a reagent to react with sortilin 1.-   58. The system of paragraph 57, wherein the isolated sample is a    blood sample.-   59. The system of paragraph 57 or 58, wherein the reagent is    selected from the group consisting of small organic or inorganic    molecules; saccharines; oligosaccharides; polysaccharides; peptides;    proteins; peptide analogs and derivatives; peptidomimetics;    glycoproteins, glycopeptides; antibodies and antigen binding    fragments thereof; nucleic acids; nucleic acid analogs and    derivatives; lipids, an extract made from biological materials such    as bacteria, plants, fungi, or animal cells; animal tissues;    naturally occurring or synthetic compositions; and any combinations    thereof.-   60. The system of any of paragraphs 57-59, wherein the reagent is a    substrate that sortilin 1 acts on, a molecule that binds with    sortilin 1, a molecule that inhibits binding of sortilin to a second    molecule; and any combinations thereof-   61. The system of any of paragraphs 57-60, wherein the reagent    further comprises a label to produce a signal so as to detect the    elevated level of the sortilin 1 in the isolated sample.-   62. The system of any of paragraphs 57-61, wherein the label is any    one or more of a radiolabel, a chromophore, a fluorophore, a    chemiluminescent precursor, a chemiluminescent reactants, or a    combination thereof.-   63. The system of any of paragraphs 57-62, wherein the elevated    level is at least 10% higher than a control or reference level.-   64. A computer system for obtaining data from at least one test    sample obtained from at least one subject, the system comprising:    -   (a) a determination module configured to receive at least one        test sample from the subject and perform at least one analysis        on at least one test sample to determine the level or amount of        sortilin 1 or a nucleic acid encoding sortiline 1;    -   (b) a storage device configured to store data output from the        determination module; and    -   (c) a display module for displaying a content based in part on        the data output from the determination module, wherein the        content comprises a signal indicative of the amount or level of        sortilin 1, and optionally the presence or absence of        calcification or CRD.-   65. A computer readable medium having computer readable instructions    recorded thereon to define software modules for implementing a    method on a computer, said computer readable storage medium    comprising:    -   (a) instructions for comparing the data stored on a storage        device with reference data to provide a comparison result,        wherein the comparison identifies elevated levels of sortilin 1;        and    -   (b) instructions for displaying a content based in part on the        data output from the determination module, wherein the content        comprises a signal indicative of elevated level of sortilin 1,        and optionally the presence or absence of calcification or CRD.-   66. The method of any of paragraphs 1-21, wherein the compound    decreases phosphorylation of sortilin 1.-   67. The method of any of paragraphs 1-21 or 67, wherein said    phosphorylation of sortilin 1 is at serine 819 or/and 825.-   68. The method of any of paragraphs 22-43, wherein the compound    decreases phosphorylation of sortilin 1.-   69. The method of any of paragraphs 22-43 or 68, wherein said    phosphorylation of sortilin 1 is at serine 819 or/and 825

Some Selected Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±5% of the value being referred to. For example, about 100 meansfrom 95 to 105.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

As used interchangeably herein, the terms “essentially” and“substantially” means a proportion of at least about 60%, or preferablyat least about 70% or at least about 80%, or at least about 90%, atleast about 95%, at least about 97% or at least about 99% or more, orany integer between 70% and 100%. In some embodiments, the term“essentially” means a proportion of at least about 90%, at least about95%, at least about 98%, at least about 99% or more, or any integerbetween 90% and 100%. In some embodiments, the term “essentially” caninclude 100%.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviations (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient” and “subject” areused interchangeably herein.

As used herein, the term “subject” is intended to mean a human or othermammal, exhibiting, or at risk of developing, calcification. Such anindividual can have, or be at risk of developing, for example, vascularcalcification associated with conditions such as atherosclerosis,stenosis, restenosis, renal failure, diabetes, prosthesis implantation,tissue injury or age-related vascular disease. The prognostic andclinical indications of these conditions are known in the art. Anindividual treated by a method of the invention can have a systemicmineral imbalance associated with, for example, diabetes, chronic kidneydisease, renal failure, kidney transplantation or kidney dialysis.

In some embodiments, a subject with mineral imbalance andcalcium/phosphate disorders, including chronic renal disease, chronicrenal failure, hemodyalysis, or diabetes suffers from acceleratedvascular and valvular calcification. In these subjects, vascular grafts,including but not limited to arterio-venous grafts/shunts forhemodyalysis access, vein grafts for occlusive peripheral arterialdisease, and saphenous vein bypass grafts for occlusive coronaryarteries, are often occluded within a year. In addition, subjects withPaget's disease, rheumatoid arthritis, osteoporosis or osteoarthritisare also at risk for calcification. Without wishing to be bound by atheory, tissue engineered vascular and valvular implants in thesepatients at risk are expected to fail in a short period of time.

In some embodiments, the subject has severe renal failure or the subjecthas a transcatheter aortic valve. The renal failure can be onhemodialysis, hemodialysis AV shunts, vein grafts, vascular anastomsis,Paget's disease, or osteoarthritis. Without limitations, these disordershave high rates or acute calcific changes, which can make measuring theeffect of the methods described herein easier.

The subject can be initially diagnosed by a licensed physician and/orauthorized medical practitioner, and a regimen for prophylactic and/ortherapeutic treatment via a method described herein can be suggested,recommended or prescribed. Thus, in some embodiments, the methodcomprises selecting a subject for treatment for cardiovascularcalcification.

Animal models that are reliable indicators of human atherosclerosis,renal failure, hyperphosphatemia, diabetes, age-related vascularcalcification and other conditions associated with cardiovascularcalcification are known in the art. For example, an experimental modelof calcification of the vessel wall is described by Yamaguchi et al.,Exp. Path. 25: 185-190, 1984, content of which is incorporated herein byreference in its entirety.

By “treatment, prevention or amelioration” is meant delaying orpreventing the onset of a disorder or reversing, alleviating,ameliorating, inhibiting, slowing down or stopping the progression,aggravation or deterioration the progression or severity of a condition.In some embodiments, at least one symptom is alleviated by at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95% but not 100%, i.e. not acomplete alleviation. In some embodiments, at least one symptom iscompletely alleviated.

As used herein, the terms “inhibiting,” “decreasing,” “preventing,” and“treating” in connection with cardiovascular calcification, are intendedto mean preventing, retarding, or reversing formation, growth ordeposition of extracellular matrix hydroxyapatite crystal deposits.Without limitations, the improvement in disorder severity includes thereversal of cardiovascular calcification, as well as slowing down theprogression of vascular calcification.

As used herein, the terms “proteins” and “peptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and “peptide”,which are used interchangeably herein, refer to a polymer of proteinamino acids, including modified amino acids (e.g., phosphorylated,glycated, etc.) and amino acid analogs, regardless of its size orfunction. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “peptide” as used herein refers to peptides, polypeptides, proteinsand fragments of proteins, unless otherwise noted. The terms “protein”and “peptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary peptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

As used herein, the term “peptidomimetic” refers to a molecule thatfolds into or has a defined three-dimensional structure similar to anatural peptide.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer specificityto sortilin 1. A full-length heavy chain includes a variable regiondomain, VH, and three constant region domains, CH1, CH2, and CH3. The VHdomain is at the amino-terminus of the polypeptide, and the CH3 domainis at the carboxyl-terminus.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer specificityto sortilin 1. A full-length light chain includes a variable regiondomain, VL, and a constant region domain, CL. Like the heavy chain, thevariable region domain of the light chain is at the amino-terminus ofthe polypeptide.

A “Fab fragment” is comprised of one light chain and the CHI andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the CH1 and CH2 domains,such that an interchain disulfide bond can be formed between two heavychains to form a F(ab′)2 molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the CH1 and CH2domains, such that an interchain disulfide bond is formed between twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

As used herein, the term “Complementarity Determining Regions” (CDRs;i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203 (hereby incorporated by reference).

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The expression “single-chain Fv” or “scFv” antibody fragments, as usedherein, is intended to mean antibody fragments that comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. (The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994)).

The term “diabodies,” as used herein, refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (VH) Connected to a light-chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger etah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol.79:315-321; Kostelny et al. (1992), J. Immunol. 148:15471553.

As used herein, the term “humanized antibody” means an antibody in whichat least a portion of non-human sequences are replaced with humansequences. Examples of how to make humanized antibodies can be found,for example, in U.S. Pat. No. 6,054,297; No. 5,886,152; and 5,877,293,content of all of which is incorporated herein by reference in itsentirety.

As used herein, the term “chimeric antibody” means an antibody thatcontains one or more regions from a first antibody and one or moreregions from at least one other antibody. The first antibody and theadditional antibodies can be from the same or different species.

As used herein, the term “antigens” refers to a molecule or a portion ofa molecule capable of being bound by a selective binding agent, such asan antibody, and additionally capable of being used in an animal toelicit the production of antibodies capable of binding to an epitope ofthat antigen. An antigen may have one or more epitopes. The term“antigen” can also refer to a molecule capable of being bound by anantibody.

As used herein, the term “polysaccharide” refers to macromolecularcarbohydrates whose molecule consists of a large number ofmonosaccharide molecules which are joined to one another by glycosidiclinkage. The term polysaccharide is also intended to embrace anoligosaccharide. The polysaccharide can be homopolysaccharides orheteropolysaccharides. Whereas the homopolysaccharides contain only onekind of unit, the heteropolysaccharides consist of monomer units ofdifferent kinds.

The term “antisense oligonucleotide” refers to single stranded DNA orRNA that is complementary to a chosen sequence. In the case of antisenseRNA, they prevent protein translation of messenger RNA strands bybinding to them. Antisense DNA can be used to target a specific,complementary (coding or non-coding) RNA. If binding takes places thisDNA/RNA hybrid can be degraded by the enzyme RNase H. Antisenseoligonucleotides are generally from to 30, from 15 to 35, or from 18 to25 nucleotides in length.

The term “shRNA” as used herein refers to short hairpin RNA whichfunctions as RNAi and/or siRNA species but differs in that shRNA speciesare double stranded hairpin-like structure for increased stability. Theterm “RNAi” as used herein refers to interfering RNA, or RNAinterference molecules are nucleic acid molecules or analogues thereoffor example RNA-based molecules that inhibit gene expression. RNAirefers to a means of selective post-transcriptional gene silencing. RNAican result in the destruction of specific mRNA, or prevents theprocessing or translation of RNA, such as mRNA.

As used herein, the term “aptamer” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules. Accordingly,aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9(1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers can be RNA or DNA based. Methods for selecting aptamersfor binding to a molecule are widely known in the art and easilyaccessible to one of ordinary skill in the art. Generally, aptamers areengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. The aptamer can be prepared by any known method, includingsynthetic, recombinant, and purification methods, and can be used aloneor in combination with other aptamers specific for the same target.Further, as described more fully herein, the term “aptamer” specificallyincludes “secondary aptamers” containing a consensus sequence derivedfrom comparing two or more known aptamers to a given target. Aptamerscan include, without limitation, defined sequence segments and sequencescomprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and non-nucleotide residues, groupsor bridges. In some embodiments, the aptamer recognizes thenon-oligonucleotide molecule or group of molecules by a mechanism otherthan Watson-Crick base pairing or triplex formation.

As used herein, the term “ribozyme” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence having specific catalytic domains that possess endonucleaseactivity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20).At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. In general, enzymatic nucleic acids act by firstbinding to a target RNA. Such binding occurs through the target bindingportion of an enzymatic nucleic acid which is held in close proximity toan enzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

Methods of producing a ribozyme targeted to any target sequence areknown in the art. Ribozymes can be designed as described in Int. Pat.Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595,each specifically incorporated herein by reference, and synthesized tobe tested in vitro and in vivo, as described therein.

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to up-regulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides can be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, content of which isincorporated herein by reference in its entirety.

As used herein, the term “calcimimetic compound” refers to a compoundthat binds to calcium sensing receptors and induces a conformationalchange that reduces the threshold for calcium sensing receptorsactivation by the endogenous ligand Ca²⁺, thereby reducing parathyroidhormone (PTH) secretion. These calcimimetic compounds can also beconsidered allosteric modulators of the calcium receptors.

Exemplary calcimimetic compounds include, but are not limited to, thosedisclosed in, for example, European Patent No. 933 354 and 1 235 797;International Publication Nos. WO 01/34562, WO 93/04373, WO 94/18959, WO95/11221, WO 96/12697, WO 97/41090; U.S. Pat. Nos. 5,688,938, 5,763,569,5,962,314, 5,981,599, 6,001,884, 6,011,068, 6,031,003, 6,172,091,6,211,244, 6,313,146, 6,342,532, 6,362,231, 6,432,656, 6,710,088,6,908,935 and U.S. Patent Application Publication No. 2002/0107406,content of all of which is incorporated herein by reference in itsentirety.

The term “fluorophore” used herein means a functional group and/or amolecule containing said functional group which will absorb energy of aspecific wavelength and re-emit energy at a different wavelength.Typically, the fluorophore is an aromatic or heteroaromatic compound andcan be a pyrene, anthracene, naphthalene, acridine, stilbene, indole,benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine,salicylate, anthranilate, coumarin, fluorescein, rhodamine or other likecompound. Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine(5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine (HAT);5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin(Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™;Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™;Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™;Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S;AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine;ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine;BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); BG-647;Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3;Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589;Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676;Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR;Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP;Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF;Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX);Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP—Cyan FluorescentProtein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA;Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp;Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; CoelenterazineO; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™;Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor(FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; DansylChloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2;Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR(Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA(4-Di-16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR;DiR (DiICl8(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP;ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidiumhomodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium;EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645; FlazoOrange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wildtype, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP);GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258;Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent compounds areavailable and can be used.

Other exemplary labels include radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C or³²P), enzymes (e.g., galactosidases, glucorinidases, phosphatases (e.g.,alkaline phosphatase), peroxidases (e.g., horseradish peroxidase), andcholinesterases), and calorimetric labels such as colloidal gold orcolored glass or plastic (e.g., polystyrene, polypropylene, and latex)beads. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and4,366,241, each of which is incorporated herein by reference in itsentirety.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photo-detector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with an enzyme substrate anddetecting the reaction product produced by the action of the enzyme onthe enzyme substrate, and calorimetric labels can be detected byvisualizing the colored label.

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

A “radiolabel” is a product that has a radioactive substance, orradionuclide incorporated in it.

The term chemiluminescence, chemiluminescent and the like refers to theproduction of light by way of a chemical reaction. It can further bedefined as the light emitted during the time that electronically excitedproducts of chemical reactions return to the ground state.

The disclosure is further illustrated by the following examples whichshould not be construed as limiting. The examples are illustrative only,and are not intended to limit, in any manner, any of the aspectsdescribed herein. The following examples do not in any way limit theinvention.

EXAMPLES Example 1: Sortilin 1 is a Novel Inducer of VascularCalcification

Vascular calcification is a prominent feature of chronic inflammatorydisorders such as chronic renal disease (CRD), diabetes mellitus andatherosclerosis, which are associated with significant morbidity andmortality. Numerous clinical (Hyder et al., American journal ofepidemiology. 2009; 169:186-194), histological (Liberman et al.,Arteriosclerosis, thrombosis, and vascular biology. 2008; 28:463-470),and animal (Bucay et al., Genes & development. 1998; 12:1260-1268 andOkayasu et al., Okayasu et al. The Journal of biological chemistry.2012; 287:19229-19241) studies suggest that processes in vascularcalcification are similar to those of bone remodeling (Khosla S. Naturemedicine. 2011; 17:430-431). Vascular calcification is an active,cell-regulated process in which vascular smooth muscle cells (SMCs) canlose the expression of their marker genes, acquire osteogenic markers,and deposit a mineralized bone-like matrix (Bostrom K I, Rajamannan N M,and Towler D A, Circulation research. 2011; 109:564-577). SMCs may playan important role in this process via transition toward anosteoblast-like state or release of calcifying matrix vesicles orapoptotic bodies. However the mechanisms of calcification remain largelyunknown. Hence, a better understanding of molecular mechanisms ofvascular calcification may lead to development of more efficienttherapeutic strategies.

Genome wide association studies (GWAS) are powerful tools to identifynovel genetic loci associated with cardiovascular disease. GWAS havestrongly associated the 1p13 locus harbouring the SORT1 gene thatencodes sortilin 1 with plasma low-density lipoprotein (LDL) cholesterollevels (Musunuru et al., Nature. 2010; 466:714-719), the onset ofmyocardial infarction (Kathiresan et al., Nature genetics. 2009;41:334-341), and coronary artery calcification (O'Donnell et al.,Circulation. 2011; 124:2855-2864). Sortilin 1 is a multi-ligand sortingreceptor with functional characteristics of the vacular protein sorting10 protein (Vps10p) domain family (Mazella et al., The Journal ofbiological chemistry. 1998; 273:26273-26276). The receptor is found inthe trans-Golgi network or early endosomes and involved inGolgi-lysosome and plasma membrane-lysosome protein trafficking (WillnowT E, Petersen C M, and Nykjaer A. Nature reviews. Neuroscience. 2008;9:899-909). Sortilin 1 is synthesized as a propeptide. Theconvertase-mediated cleavage in the late trans-Golgi activates itsspecialized tissue-specific ligand sorting roles (Munck et al., The EMBOjournal. 1999; 18:595-604). Evidence suggests the binding of Sortilin 1to lipoprotein lipases (Nielsen et al., The Journal of biologicalchemistry. 1999; 274:8832-8836), apolipoprotein (Apo) AV (Nilsson etal., The Journal of biological chemistry. 2008; 283:25920-2592), andApoB100 (Musunuru et al., Nature. 2010; 466:714-719 and Kjolby et al.,Cell metabolism. 2010; 12:213-223). Two recent studies reported thefunction of SORT1 in lipid metabolism. Musunuru et al. provided theevidence that high sortilin 1 levels are associated with reduced hepaticvery low density lipoprotein (VLDL) secretion and lower LDL cholesterollevel using a humanized mouse model with liver specific sortilin 1deletion or overexpression (Musunuru et al., Nature. 2010; 466:714-719).In contrast, Kjolby et al. showed that the global absence of Sort1reduces secretion of lipoproteins from the liver and ameliorateshypercholesterolemia and atherosclerosis in LDL receptor-deficient mice(Kjolby et al., Cell metabolism. 2010; 12:213-223). These contradictoryfindings related to lipid metabolism remain a subject of debate.

Although GWAS linked the SORT1 gene to coronary artery calcification,and genetic deletion of SORT1 in mice reduced atherosclerotic lesionformation, the role of sortilin 1 in processes of vascular calcificationis utterly unknown. Data presented herein demonstrates for the firsttime that sortilin 1 contributes to the pathogenesis of vascularcalcification.

Materials and Methods

Cell Culture:

Human coronary artery smooth muscle cells (HCASMCs) were purchased fromPromocell (Heidelberg, Germany) and were grown in Smooth Muscle CellGrowth Medium 2 (SMC-GM2, Promocell) supplemented with epidermal growthfactor (0.5 ng/ml), insulin (5 μg/ml), basic fibroblast growth factor-B(2 ng/ml) and fetal bovine serum (5%). The cells were maintained at 37°C. (5% CO2, 90% humidity) and were used between passages 3 and 8. Cellsisolated from three to four independent donors were used.

Human Tissue:

Atherosclerotic carotid arteries (n=20) were obtained from patientsundergoing endarterectomy surgery at Brigham and Women's Hospitalaccording to IRB protocol, Samples were embedded in OCT compound andstored at −80 degree until use.

Animal Procedure:

30-week-old Apoe−/− mice (Jackson Laboratory, Bar Harbor, Me., USA)consumed an atherogenic diet (D12079B; 41% milk fat, 0.2% totalcholesterol, Research Diet, New Brunswick, N.J., USA) from 10 weeks ofage. At 20 weeks of age, mice in each group were randomized either tocontinue with the atherogenic high-cholesterol diet or a CRD group. Weused a two-step procedure to create CRD as previously described inAikawa et al., Circulation. 2009; 119:1785-1794. Briefly, one week aftera left hemi-nephrectomy a right total nephrectomy was performed. At 10weeks after surgery, mice were sacrificed for correlative histologicalanalyses. The Institutional Animal Care & Use Committee at Beth IsraelDeaconess Medical Center approved all procedures (protocol #017-2010).Age-matched wild-type C57/BL6 mice (Jackson Laboratory) served ascontrol.

Osteogenic Transition of Human Vascular SMCs:

SMCs were cultured for up to 21 days in the presence of either controlmedium (DMFM, 10% FBS, 1% antibiotics) or osteogenic medium, whichconsisted of control medium supplemented with 10 nmol/l dexamethasone,10 mmol/l β-glycerol phosphate, and 100 mmol/1 L-ascorbate phosphate.Medium was changed three times per week. Recombinant sortilin 1 (200ng/ml; BioVendor, Candler, N.C., US) was replaced at every mediumchange.

RNA Interference of Sortilin 1:

RNA silencing of sortilin 1 was performed as described previously inGoettsch et al., Endocrinology. 2011; 152:4915-4926. Briefly, 50 nmol/lsiSORT1 (ONTARGETplus SMART-pool (L-010620), Thermo Scientific,Lafayette, Colo., USA) or non-targeting siRNA (ON-TARGET Non-TargetingPool, Thermo Scientific) was transferred into SMCs using Dharmafect 1(Dharmacon RNAi Technologies). Transfection was performed twice per weekover the entire cell culture period.

Adenoviral Overexpression of Sortilin1:

Human SORT1 (NM_002959.4) ORFEXPRESS™ Gateway® PLUS Shuttle Clone waspurchased from GeneCopoeia (Rockville, Md., USA). The ViralPowerAdenoviral Expression System together with pAd/CMV/V5-DEST asdestination vector was used (Life Technologies, Grand Island, MY, USA).Recombination reaction between attL and attR sites was performed usingLR Clonase II enzyme mix resulting in pAd/CMV/SORT1. pAd/CMVN5-GW/lacZwas used as control vector. Adenovirus was amplified by transfection ofPacI-digested vector in HEK293A cells according to the manufacturesprotocol. Multiplicity of infection (MOI) was determined by Adeno-XRapid Titer Kit (Clontech, Mountain View, Calif., USA). SMCs weretransduced with sortilin 1 and control (LacZ) adenoviruses at MOI of100. For long term cell culture transduction was repeated every 7 days.

Cell Viability:

Cell viability was assessed using the Cell Titer Blue assay (Promega,Heidelberg, Germany) according to the manufacturer's protocol.

miRNA Inhibition and Induction:

Inhibition and promotion of miRNA was performed as previously describedin Goettsch et al., The American journal of pathology. 2011;179:1594-1600. To inhibit the function of miR-125b, a miR inhibitor(antimiR-125b; Life Technologies) and a negative control (anti-negativecontrol; Life Technologies) were used. A miR precursor (pre-miR-125b;Life Technologies) and a negative control (pre-negative control; LifeTechnologies) were used to promote the function of miR-125b.Transfection of 50 nmol/L RNAs was performed by lipofection using siPORTNeoFX Transfection Agent (Life Technologies).

3′-UTR Luciferase Binding Assay:

Human SORT1 (HmiT016537-MT01) and control (CmiT000001-MT01) 3′-UTRluciferase constructs were purchased from GeneCopoeia. Transienttransfection of luciferase vector in SMCs was conducted using Fugene HD(Roche). In parallel, cells were transfected with 50 nmol/L miRNAprecursor, inhibitor, or the corresponding controls as described in themethod section. The luciferase signal was measured using Luc-Pair miRLuciferase assay (GeneCopoeia) 48 h after transfection. Fireflyluciferase was normalized with Renilla luciferase in the same well.

Immunohistochemistry/Immunofluorescence:

Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min.Cells were permeabilized for 10 min in 0.5% Triton X-100 andsubsequently washed and blocked with 1% BSA in PBS for 30 min. The glassslides were exposed to anti-Sortilin 1 (1:100; BD, San Diego, Calif.,USA) and anti-Osteopontin (OPN) (1:100; Abcam, Cambridge, Mass., USA)antibody for 2 hours and then incubated with an Alexa Fluor 488-labelledsecondary antibody and Alexa Fluor 594-labelled secondary antibody (LifeTechnologies). After three washing steps, nuclear staining with DAPI wasperformed and slides were covered using a mounting medium (Dako,Glostrup, Denmark).

Tissue was cut into 7 μm-thin slices, and cryo-sections were fixed inacetone. After blocking in 4% of appropriate serum, sections wereincubated with primary antibody [human SORT1 (1:100; BD) or mouse SORT1(1:100; R&D systems, Minneapolis, Minn., USA)], followed bybiotin-labelled secondary antibody (Vector Laboratories, Burlingame,Calif., USA) and streptavidin-coupled Alexa Fluor 488 antibody (LifeTechnologies). For immunofluorescence double labelling: afteravidin/biotin blocking (Vector Laboratories), the second primaryantibody [RUNX2 (Novus, St. Charles, Mo., USA) or OPN (Abcam)] wereapplied overnight at 4 degrees followed by biotin-labelled secondaryantibody and streptavidin-coupled Alexa Fluor 594 antibody (LifeTechnologies). Sections were washed in PBS and embedded in mountingmedium containing DAPI.

Bright field immunohistochemistry on tissue sections: after firstbiotin-labelled secondary antibody, tissue sections were incubated withstreptavidin-labelled HRP solution (Dako) followed by AEC solution.

Slides were examined using the Eclipse 80i microscope (Nikon, Melville,VY) or the confocal microscope A1 (Nikon). All images were processedwith the Elements 3.20 software (Nikon).

RNA Preparation and Real-Time PCR:

Total RNA from the cell culture was isolated using the TriZol (LifeTechnologies) and reverse transcription was performed using theQuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The mRNAexpression was determined by TaqMan-based real-time PCR reactions (LifeTechnologies). The following TaqMan probes were used: Hs00361747_ml(human SORT 1), Hs01011692_ml (human PHEX), Hs01047978_ml (human RUNX2),and 4326315E (human β-actin). The expression levels were normalized toβ-actin. The results were calculated using the ΔΔCt method, andpresented in fold increases relative to control. Human Osteogenesis RT²Profiler™ PCR Array (Qiagen) was used to profiles the expression of 84genes related to osteogenic differentiation.

Western Blot Analysis:

Cells were lyzed with RIPA buffer containing protease inhibitor (Roche).Protein concentration was measured using BCA method (Thermo Scientific).Total protein was separated by 8-12% SDS-PAGE and transferred using theiBlot Western blotting system (Life Technologies). Primary antibodiesagainst human SORT1 (1:1,000; Abcam or 1:200; BD), human PHEX (1:200;OriGene, Rockville, Md., USA) and human β-actin (1:5,000; Novus) wereused. Protein expression was detected using Pierce ECL Western Blottingsubstrate Reagent (Thermo Scientific) and ImageQuant LAS 4000 (GEHealthcare, Waukesha, Wis., USA).

Mineralization Assay and Activity of Tissue Non-Specific AlkalinePhosphatase:

Mineralized matrix formation was assessed by Alizarin Red S staining.SMCs were fixed in 4% PFA and stained with 40 mM Alizarin Red S (pH 4.2)for 30 min at RT. Excess dye was removed by washing the plates withdistilled water.

Accumulated calcium in the mineralized matrix was eluted using 0.6Nhypochloric acid and determined using the Calcium Colorimetric Assay Kit(BioVision, Milpitas, Calif., USA). Tissue non-specific alkalinephosphatase (TNAP) activity was measured in cell cultures using theAlkaline Phosphatase Activity Colorimetric Assay Kit (BioVision).

Collagen Assay:

Collagen content was determined in the extracellular matrix using SircolSoluble Collagen Assay (Biocolor, Northern Ireland, UK). Briefly, aftera washing step, cells were incubated with 0.5 M acetic acid/0.1 mg/mlpepsin overnight. After neutralization, collagen was concentratedovernight. The next day, Sircol reagent was added to bind collagen.After two washing steps the Sircol dye was released from thecollagen-dye complex by alkali reagent and absorption at 555 nm wasmeasured.

Measurement of Serum Sortilin 1 and FGF23:

Serum sortilin 1 and FGF23 were measured by enzyme-linked immunosorbentassay (ELISA) according to the manufacturer's instructions. Serumsortilin 1 was analyzed using an immunoassay from Cusobio (Wuhan, China)and FGF23 was determined using an immunoassay from Millipore (Billerica,Mass., USA).

Co-Immunoprecipitation:

Cells were lyzed in immunoprecipitation (IP) lysis buffer (ThermoScientific). Sortilin 1 antibody (5 μg, Abcam) or IgG rabbit controlantibody (5 μg, Santa Cruz Biotechnology, Santa Cruz, Calif., USA) wereincubated with Dynabeads Protein G (Life Technologies) by a two hourrotating incubation at 4 degrees followed by 3 times washing withPBS/Tween 20 (0.002%) using a magnet. One mg of protein was pre-clearedby incubation with the bead-bound IgG antibody for one hour at 4 degreesunder rotating conditions. Non-IgG-bound protein was divided andtransferred to either bead-bound sortilin 1 antibody or bead-bound IgGantibody, and was incubated for four hours at 4 degrees under rotatingconditions. Bead-antibody-protein complex was washed 3 times in 1,000 μlwashing buffer (PBS). Ninety percent of the IPed protein sample wassubjected to SDS-PAGE and visualized by coomassie stain (BioRad,Hercules, Calif., USA). The prominent band corresponding to the expectedmolecular weight for SORT1 (corroborated by a parallel Western blotanalysis) was excised for in-gel trypsinization. The remaining gel lanefor each co-IP condition was divided into 8 bands, and in-geltrypsinized. Peptides were dissolved in 20 μl sample loading buffer(0.1% formic acid, 5% acetonitrile) for subsequent mass spectrometricanalysis.

Mass Spectrometry:

Peptide samples were analyzed with the high resolution/accuracyLTQ-Orbitrap (Elite model) mass spectrometer fronted with a NanosprayFLEX ion source, and coupled to an Easy-nLC1000 HPLC pump (ThermoScientific). The peptides were subjected to a dual column set-up: anAcclaim PepMap RSLC C18 trap column, 75 μm×20 mm; and an Acclaim PepMapRSLC C18 analytical column 50 μm×150 mm (Thermo Scientific). Theanalytical gradient was run at 250 nl/min from 10 to 30% Solvent B(acetonitrile/0.1% formic acid) for 30 minutes, followed by five minutesof 95% Solvent B. Solvent A was 0.1% formic acid. All reagents wereHPLC-grade. The instrument was set at 120 K resolution, and the top 20precursor ions (within a scan range of 380-2000 m/z) were subjected tocollision induced dissociation (collision energy 35%) for peptidesequencing (MS/MS). The dynamic exclusion feature was disabled.

Analysis of Mass Spectrometry Data:

The MS/MS data were queried against the Human UniProt database(downloaded on May 27, 2012) using the SEQUEST search algorithm (Yateset al., Analytical chemistry. 1995; 67:1426-1436) via the ProteomeDiscoverer (PD) Package (Thermo Scientific) using methionine oxidationas a variable modification, and carbamidomethylation of cysteineresidues as a fixed modification. The peptide false discovery rate (FDR)was calculated using Percolator provided by PD: the FDR was determinedbased on the number of MS/MS spectral hits when searched against thereverse, decoy Human database (Elias J E and Gygi S P, Nature methods.2007; 4:207-214 and Kall et al., Journal of proteome research. 2008;7:29-34). Peptides were filtered based on a 1% FDR, and proteins withthree or more unique peptides were analyzed.

Sortilin 1-specific proteins were differentiated from non-specificproteins by subtracting the protein hits derived from IgG controls fromthose of the corresponding sortilin 1 IPs. The final co-IP lists includeproteins that are completely absent in the IgG samples. For proteinsthat were present in both SORT1 and IgG IPs but may have been enricheddue to SORT1 co-IP, only those with peptide-spectrum matches (PSMs)(Stevenson et al., Journal of proteomics. 2009; 72:555-566) 20-fold orgreater (with respect to the IgG control) were included in the analysis.The PSM cut-off was established by the observed PSM counts of the myosinbands in FIG. 6A.

Statistical Analysis:

Data are given as means±SD, and n indicates the number of independentexperiments. Statistical analyses were performed using a one-way ANOVAwith Bonferroni's post hoc test, and single group comparisons using aStudent's t test. Correlation analyses were performed according toPearson. A value of p<0.05 was considered statistically significant.

Results and Discussion

Sortilin 1 is Expressed in Human Calcified Lesions and Induced DuringOsteogenic Transition of Human Vascular SMCs.

Staining of calcified human carotid endarterectomy samples indicated ahigh expression of sortilin 1 in calcified regions (FIG. 1A). Cells inthese areas showed a weak alpha smooth muscle actin (αSMA) staining andwere negative for the macrophage marker CD68 (FIG. 1A). Due to the lossof SMC marker genes during osteogenesis, sections were stained forRunx2/Cbfal, a marker of osteogenic transition. Indeed, in humancalcified lesion, sortilin 1 was highly expressed in cells withactivated, nucleus-located Runx2 (FIG. 1B).

We subsequently investigated whether sortilin 1 expression is involvedin the process of vessel calcification. Human coronary aortic SMCs(HCASMCs) cultured in osteogenic medium (OM) induced sortilin 1expression in a time-dependent manner. Calcified SMCs (Day 21) showed15-fold and 22-fold increases in sortilin 1 mRNA and protein expression,respectively, as compared to day 1, and a 2.5-fold induction compared tocells cultured for 21 day in control medium (FIGS. 1C and 1D).Immunofluorescence staining co-localized high sortilin 1immunoreactivity with that of high osteopontin (OPN) immunoreactivity,an osteogenic marker for calcified SMCs (FIG. 1E).

Sortilin 1 Directly Affects Matrix Mineralization of Human VascularSMCs.

To demonstrate the direct role of sortilin 1 in the calcification ofSMC, we performed gain-of-function and loss of-of-function experiments.Long-term silencing of sortilin 1 by siRNA consistently suppressedsortilin 1 mRNA expression by 60-70% (FIG. 7A) and protein expressionafter 21 days (FIG. 7B).

Silencing of sortilin 1 caused a significant reduction of TNAP activity(−30%, p=0.009) in calcified SMCs (FIG. 2A), whereas increased exogenoussortilin 1 levels using recombinant sortilin 1 promoted TNAP activity by37% (p=0.016, FIG. 2B). Moreover sortilin 1 silencing decreased noduleformation (FIG. 2C) demonstrated by a 33% reduction of the mineralizedmatrix (p=0.004, FIG. 2D). In contrast, elevated exogenous sortilin 1level enhanced the matrix mineralization by 77% (p=0.002, FIG. 2E).These data were further supported by increasing endogenous sortilin 1level using adenoviral overexpression. Infection of SMCs with adenoviralSortilin 1 resulted in a 2-fold increase in TNAP activity (p=0.015, FIG.2F) and enhanced matrix mineralization by 30% (p=0.028, FIG. 2G)compared to adenoviral LacZ control.

Of note, modulation of sortilin 1 had no effect on collagen accumulationand secretion. The matrix collagen content of calcified SMCs (FIGS. 10Aand 10B) and released collagen (data not shown) did not alter uponsortilin 1 silencing or increased exogenous sortilin 1.

Collectively, these results demonstrate that sortilin 1 promotes matrixmineralization and TNAP activity in calcified human vascular SMCs.

Sortilin 1 Modulates PHEX in Calcifying Human Vascular SMCs.

We then investigated the mechanisms behind sortilin 1 action. First weanalyzed cell viability, which was unaffected by sortilin 1 silencing,overexpression and stimulation (FIG. 3A). Unexpectedly, the mainosteogenic transcription factor Runx2 was significantly repressed bysortilin 1 silencing, but was not affected by stimulation with sortilin1 (FIG. 10C). Consequently, we analyzed the expression of 84 transcriptsrelated to osteogenesis using a PCR array in calcified SMCs with eithersilenced sortilin 1 or stimulation with recombinant sortilin 1. We foundthat PHEX, a phosphate regulating endopeptidase, was 1.5-fold induced bysortilin 1 silencing. In contrast, PHEX expression decreased by 77% withincreased exogenous sortilin 1 (FIG. 3B). We further validate these datausing TaqMan real time PCR and Western blot analysis. PHEX expressionwas not affected by either the silencing or the recombinant expressionof sortilin 1 in SMCs cultured in control medium (FIG. 3C). However,calcified SMCs showed strong inhibition of PHEX, which was significantlyrecovered after sortilin 1 silencing. Stimulation with sortilin 1 undercalcifying condition decreased PHEX mRNA (FIG. 3D). The possibleinterplay between PHEX and sortilin 1 was further supported by astatistically significant negative correlation (r=−0.865; R²=0.748;p=0.0001; FIG. 3E).

Sortilin 1 is Repressed by miR-125b.

We further examined what regulates sortilin 1 expression in the contextof SMC calcification. Our previous studies demonstrated repression ofmiR-125b in calcified human vascular SMCs (Goettsch et al., The Americanjournal of pathology. 2011; 179:1594-1600). In silico analyses using 3different databases (www.targetscan.com, www.microRNA.org,http://pictar.mdc-berlin.de) revealed that miR-125b, a highly conservedmiRNA, may suppress sortilin 1 expression by binding the 3′UTR (FIG.4A). Treatment with osteogenic medium suppressed miR-125b expression(−60%) in SMCs (FIG. 4B), which is consistent with increased sortilin 1expression levels as shown in FIGS. 1C and 1D. To determine the role ofmiR-125b in sortilin 1 expression, we then performed loss-of-functionand gain-of-function experiments. Inhibition of miR-125b using anti-miRin the control medium caused a 2-fold increase in sortilin 1 expression(FIG. 4B), which was comparable to cells cultured in the osteogenicmedium transfected with scramble anti-miR (FIG. 4C).

In addition, sortilin 1 and miR-125b expression levels negativelycorrelated in a statistically significant manner (r=−0.825; R²=0.681;p=0.043; FIG. 4D), suggesting a direct interaction. A luciferase assaywas then devised to determine the direct binding of miR-125b to theSORT1 3′UTR. Inhibition of miR-125b promoted SORT1 3′UTR reporteractivity, whereas mimicking miR-125b repressed the activity (FIG. 4E).These lines of evidence indicate that binding of miR-125b to the 3′UTRof SORT1 indeed represses sortilin 1 expression, and suggest that thedecreased expression of miR-125b in calcified SMCs contributes to theinduction of sortilin 1 in vascular calcification.

Sortilin 1 is Increased in Chronic Renal Disease.

We further assessed vascular expression of sortilin 1 in Apoe−/− micefed a high fat diet and Apoe−/− mice with CRD, induced by 5/6nephrectomy. Of note, CRD accelerates both intimal and medialcalcification in Apoe−/− mouse model (Aikawa et al., Circulation. 2009;119:1785-1794). In the aortic media of Apoe−/− mice, sortilin 1immunopositive area was 46-fold greater as compared to wild type mice,and was further increased by 5/6 nephrectomy (6.6-fold, p=0.024) (FIGS.5A-5C). In calcified intima, induction of CRD increased sortilin 1immunopositive area by 3.6-fold (p=0.029; FIGS. 5B and 5D). These datawere confirmed on mRNA level (rel. sortilin 1 mRNA expression: Apoe−/−;0.19±0.08, CRD; 0.37±0.06; p=0.04) Sortilin 1 serum levels were elevatedin Apoe−/− mice with and without CRD by 2.7 and 3.3-fold compared towild type mice (p=0.008; FIG. 5E). Elevated serum phosphate levels, atypical feature of CRD, observed in our model (WT: 6.6±0.1 mg/dL;Apoe−/−: 20.5±1.2 mg/dL, CRD: 54.3±10.2 mg/dL, p<0.01), correlated withincreased serum sortilin 1 (r=0.831, R²=0.691, p=0.008, FIG. 5G).Furthermore, FGF23, a known regulator of phosphate homeostasis was4-fold higher in serum of CRD mice as compared to wild type mice (FIG.5F).

We further investigated whether high phosphate levels could triggerSORT1 activation. Stimulation of SMCs with phosphate increased sortilin1 mRNA expression in a dose-dependent manner (FIG. 10). High phosphatelevel also promoted expression of sortilin 1 protein (FIG. 10).

Identification of Key Sortilin-1 Interacting Proteins:

Sortilin 1—regulated calcification is in part dependent on PHEX, thus isit conceivable to propose that additional proteins act to either promoteor inhibit sortilin 1 activity via direct physical associations. Wetherefore sought to investigate potential binding partners of sortilin 1by performing co-immunoprecipitation (co-IP) of sortilin 1 from humanvascular SMCs cultured in either control media or osteogenic/calcifyingmedia, and by using high resolution mass spectrometry (MS) as theanalytical method.

The Coomassie stained SDS-PAGE gel in FIG. 6A represents the sortilin 1interactome from one out of three donor. We identified approximately 150proteins that co-IP with sortilin 1 in both control and calcifyingconditions (FIG. 6B), the prominent bands corresponded to sortilin 1,vimentin, myosin 9, myosin 10, and α-actin (FIG. 6A). A strikingdifference in the banding pattern between the control and calcifyingconditions was the near disappearance of the myosins and α-actin (FIG.6A, with green arrow heads). We used the peptide-spectral countingmethod (Stevenson et al., Journal of proteomics. 2009; 72:555-566) tocompare the relative abundances of all 150+ proteins in control versuscalcifying conditions. FIG. 6B demonstrates the correlation between therelative band intensities observed in the SDS-PAGE gel and immunoblotdata, and the spectral count ratios of calcifying versus controlconditions: sortilin 1 levels increase in calcified SMCs, whereas themyosins and α-actin decrease, and vimentin remains constant. Our ongoinganalyses include analysis of sortilin 1 co-IPs from the same donor inhigh stringency conditions in order to differentiate between thestronger binding sortilin 1-core complex (high stringency) from thoseincluding peripheral binding proteins (low stringency). Ongoing analysisalso includes co-IPs from additional donors. Thus far, our preliminarydata implicate a number of protein trafficking interactors of sortilin1, indicating that SMC calcification depends on subcellularreorganization of the sortilin 1 protein.

Sortilin 1 Modulates PHEX in Calcifying Human Vascular SMCs:

To further proof the interplay between PHEX and sortilin 1, we performedloss-of-function studies. Silencing of PHEX did not blocksortilin-1-dependent inhibition of SMC calcification (FIG. 9). Inconclusion, the effect of sortilin 1 on PHEX expression does notdirectly involved in SMC calcification.

Sortilin 1 does not Affect Osteoblastogenesis In Vitro:

Vascular calcification and bone remodeling share common pathways.Therefore, a drug developed to treat or prevent vascular calcificationshould not affect bone remodeling. We analyzed the expression ofsortilin 1 in osteoblasts differentiated from human mesenchymal stromalcells (hMSC), precursors of bone osteoblasts. Sortilin 1 proteinexpression does not increase during osteoblastogenesis (FIG. 10A).Furthermore, silencing of sortilin 1 or increasing of exogenous sortilin1 does not alter alkaline phosphatase activity (FIG. 10B) and matrixmineralization (FIG. 10C) in human osteoblasts.

Extracellular Vesicles Contain Sortilin 1:

Based on our evidence that exogenous sortilin 1 increases matrixmineralization and serum sortilin 1 levels are elevated inatherosclerotic mice in our study, we also hypothesized that circulatingsortilin 1 acts as a ligand for unknown cell-surface receptor or as adecoy receptor, which may trigger intracellular events leading tocalcification. Indeed, shedding of luminal domain byzinc-metalloproteases and ADAM10 resulted in a soluble form of sortilin1 (Navarro V, et al. Biochemical and biophysical researchcommunications. 2002; 298:760-764).

In order to understand a from of circulating sortilin 1 (soluble vsvesicles), we analyzed cell culture supernatant as well as SMC-derivedextracellular/matrix vesicles, which also contribute to thecalcification process. We precipitated the protein from 2 ml cellculture supernatant and performed Western blot analysis. Using cellculture supernatent we detected sortilin 1 protein using adenovirussortilin 1 overexpression (FIG. 11A). Cell culture supernatant fromcontrol or calcified cells and from LacZ control cells did not showsortilin 1 protein expression. Next, we isolated extracellular vesiclesreleased from control or calcificed SMC into the cell culturesupernatant and were able to detected sortilin 1 protein inextracellular vesicles from calcified SMC.

Identification of Key Sortilin4 Interacting Proteins:

We evaluated our proteomic data. FIG. 6B shows selected binding partnersof sortilin 1, which are more or less or equally associated withsortilin 1 in calcified SMC.

We confirmed the loss of association between sortilin 1 and myosin-9using Western blot (FIG. 12). The association of sortilin 1 tocaveolin-1 (Cav-1) and tissue non-specific alkaline phosphatase (TNAP)is promoted in calcified SMC (FIG. 13). Next, we examined whether Cav-1is increased during the SMC calcification. Indeed caveolin-1 as well asTNAP were increased in calcified SMC (FIG. 14).

TABLE 1 Sortilin 1 binding partners from 3 independent experiments.Protein 1 2 3 Tropomyosin alpha-3 NM NM NM Actin, beta 0.19* NM NMRetinoic acid-induced 14 NM 0.29* NM Actin-related protein 2/3 0.63 NMNM Actin-related protein 3 NM — NM Alpha-actinin NM — NM PDZ and LIMdomain protein 4 NM — NM Myosin-9 0.1* 0.5* 0.2* Transitionalendoplasmic reticulum ATPase 0.5 1.1 1.2  Caveolin-1 5.0* 3.6* OMSortilin 1 12.8* 4.6* 8.1* V-type proton ATPase subunit C 1 OM OM —Guanine nucleotide-binding protein subunit beta-1 OM OM — Alkalinephophatase OM OM — Calpain-1 catalytic subunit — OM OM Cathepsin D 0.65OM OM Erlin-1 OM OM OM Ratio (calcified/control) of spectral counts. NM;uniquely associated with sortilin 1 in control SMC, OM; uniquelyassociated with sortilin 1 in calcified SMC, —; not detected.

Sortilin 1 is Loaded into Matrix Vesicles (MV).

Recent studies identified that SMC-induced matrix mineralization mayalso proceed through the release of calcifying MVs. Indeed, we detectedsortilin 1 in calcified MVs of human plaques using a transmissionelectron microscopy-based immunogold approach (FIG. 17A). Westernblotting MV lysates isolated from supernatant of cells cultured incontrol or osteogenic medium detected sortilin 1 in calcified conditions(FIG. 17B). Mass spectrometry also verified the presence of sortilin 1in MVs derived from both control and osteogenic conditions (FIGS. 17Cand 17D). Sortilin 1 loading into MVs increased in sortilin 1overexpressing SMCs and was further elevated in MVs derived fromcalcified SMCs as demonstrated by Western blot and massspectrometry-based parameters such as the number of peptides andpeptide-spectrum matches (PSMs) (FIG. 17D). We also determined that MVrelease is not dependent on sortilin 1 loading. The numbers of MVsreleased in control versus siRNA-targeted sortilin 1 SMCs were similarwhen analyzed by Nanosight nanoparticle tracking system (FIG. 17E).

Sortilin 1 Re-Distributes to Lipid Raft/Caveolae-Enriched Membrane inCalcified SMCs.

Sortilin 1 is known to traffic within the cell. Recent studies suggestthat lipid rafts play an essential role in regulated exocytosis pathway.To investigate if SMC calcification affects the cellular redistributionof sortilin 1, we performed a hydrodynamic method that usesdiscontinuous sucrose density gradients to resolve caveolae-enrichedmembrane/lipid rafts (CEM) from other cellular constituents notassociated with CEM (nCEM). Under control conditions, we identifiedsortilin 1 in both CEM and nCEM fractions (FIG. 18A). We assayed thesesame fractions for caveolin-1 expression, which served as a positivecontrol. Following SMC calcification, there was a redistribution ofsortilin 1 into CEM fractions, suggesting an enrichment of sortilin 1into specialized membrane domains upon SMC calcification (FIG. 18A).Calcified SMC showed increased caveolin-1 expression, which localizes inCEM (FIG. 18A) as well as caveolin-1 phosphorylation (Tyr-14) (FIG.18B). Silencing of caveolin-1 reduced sortilin 1 protein levels in wholecell lysates from calcified SMCs (FIGS. 18B and 18C), as well as in theCEM (FIG. 18C). Furthermore, silencing of caveolin-1 reduced TNAPactivity (−31%, p<0.001) and SMC calcification (−47%, p<0.001; FIGS. 18Dand 18E). SMCs isolated from caveolin-1-deficient mice revealedabolished sortilin 1-mediated induction of TNAP and calcification (FIGS.18F and 18G). We then investigated whether modulation of sortilin 1affects the calcification potential of MVs. Silencing of cellularsortilin 1 reduced TNAP activity within the MVs (−45%; p<0.001) (FIG.18H). Further, we detected caveolin-1 and TNAP in the MV as well, whichwere enriched in calcified SMCs (FIG. 17B).

Phosphorylation on S₈₂₅ and S₈₁₉ Affect Cellular ALP Activity and MatrixMineralization.

We observed an increased phosphorylation of the C-terminal intracellulardomain tail, SGYHDDpS825DEDLLE (SEQ ID NO: 10), of sortilin 1. Themutation of S₈₂₅A, S₈₂₅D, S₈₁₉A, S₈₁₉D and the deletion of theC-terminal 6 amino acids were confirmed by sequencing. The functionalityof the constructs were confirmed by Western blot. The goal was toidentify the functional consequence of the C-terminal serinephosphorylation site.

Overexpression of sortilin 1 promotes ALP activity by 2-fold. Preventionof the phosphorylation at S₈₂₅ and S₈₁₉ and the deletion of theC-terminal 6 amino acids significantly reduced ALP activity (p<0.05,n=3); whereas constitutive active phosphorylation of S₈₂₅ and S₈₁₉promoted ALP activity 0.5 fold and 2.8 fold, respectively. In line withthese findings, the sortilin 1-induced matrix mineralization wasprevented by overexpression of S₈₂₅ and S₈₁₉ as well as by the deletionof the C-terminal 6 amino acids. Overexpression of the constitutiveactive S₈₁₉ phosphorylation further promoted matrix mineralization.Results are shown in FIG. 19.

The present study identified sortilin 1 as a novel contributor tocardiovascular calcification. Key findings documented here (1) revealedthat high sortilin 1 expression co-localized with osteogenic markers invitro and in vivo in human and mouse tissues and cells; (2) demonstratedan induction in serum sortilin 1 levels and an increase in sortilin 1 inaortic media in mouse CRD model; (3) determined miR-125b as a modulatorof sortilin 1; and (4) provided mechanistic in vitro evidence for adirect role of sortilin 1 in osteogenic changes using gain-of-functionand loss-of-function studies. In addition, we demonstrated in vitro thatincreased sortilin 1 levels promote vascular calcification via aPHEX-dependent mechanism, whereas decreased sortilin 1 levels preventcalcification. Moreover, the present study provides evidence that thesortilin 1 pathway is a novel mechanism of phosphate- andmicroRNA-dependent osteogenic transition of vascular SMCs towards anosteoblastic phenotype, in a manner independent of the Runx2 pathway.

Previously, the 1p13 locus harbouring the SORT1 gene, encoding sortilin1 was associated with coronary artery disease (Samani et al., The NewEngland journal of medicine. 2007; 357:443-453), particularly coronaryartery calcification (O'Donnell et al., Circulation. 2011;124:2855-2864). Thus, the present study may provide biological andmolecular explanations for the observed link. Sortilin 1 promotes thecalcification of SMCs. Our data are in line with the evidence associatedwith reduced atherosclerotic lesions in SORT1/LDL receptor doubleknockout mice (Kjolby et al., Cell metabolism. 2010; 12:213-223).Furthermore, non-vascular sortilin 1 expression was repressed inhigh-fat diet models of obesity in a lipoprotein-dependent manner (Ai etal., The Journal of clinical investigation. 2012; 122:1677-1687 andKaddai et al., Diabetologia. 2009; 52:932-940). Using an animal model ofCRD, we observed a strong increase in vascular sortilin 1 expression aswell as elevated sortilin 1 serum levels. CRD is characterized byincreased serum phosphate levels and no alterations in the lipidprofile. Thus, the role of sortilin 1 in vascular calcification in CRDmay be partially independent from hyperlipidaemia, and enhanced withphosphatemia. In our study, 5/6 nephrectomy significantly increasedsortilin 1 expression in medial SMC in Apoe−/− mice offering a possibleexplanation for the observation that atherosclerotic plaques of patientswith CRD and cardiovascular disease have intimal calcification, whereasmedical calcification often occurred only in CRD patients (Nakamura etal., Clinical journal of the American Society of Nephrology: CJASN.2009; 4:1892-1900). Furthermore, arterial medial calcification is highlycorrelated with serum phosphate levels (El-Abbadi et al., Kidneyinternational. 2009; 75:1297-1307 and Ishimura et al., American journalof kidney diseases: the official journal of the National KidneyFoundation. 2005; 45:859-865). Indeed, we observed the induction ofsortilin 1 by phosphate, which was further supported by a significantcorrelation of serum sortilin 1 and phosphate levels in the CRD mousemodel. In addition, our study showed that sortilin 1 inhibits thephosphate-regulating endopeptidase PHEX in SMCs. The inactivatingmutations of PHEX altered the responsiveness of bone cells toextracellular phosphate concentrations which may create a lower setpoint for “normal” phosphate levels (Ichikawa et al., Journal of boneand mineral research: the official journal of the American Society forBone and Mineral Research. 2012; 27:453-460) and it stimulated FGF23gene transcription (Liu et al., American journal of physiology.Endocrinology and metabolism. 2006; 291:E38-49 and Martin et al., FASEBjournal: official publication of the Federation of American Societiesfor Experimental Biology. 2011; 25:2551-2562). In line with thisevidence, we observed elevated FGF23 levels in parallel to increasedsortilin 1 levels in CRD mice. Collectively, our results suggest thatsortilin 1 participates in hyperphosphatemia-related vascularcalcification involving the FGF23 pathway. In support to this notion, itwas shown that increased serum FGF23 serum levels are associated withabdominal aortic calcification in men (Schoppet et al., The Journal ofclinical endocrinology and metabolism. 2012; 97:E575-583) and vascularcalcification in patients with CRD (Desjardins et al., Osteoporosisinternational: a journal established as result of cooperation betweenthe European Foundation for Osteoporosis and the National OsteoporosisFoundation of the USA. 2012; 23:2017-2025).

It is well established that phosphate direct affects SMC calcificationby stimulation of osteogenic/chondrogenic differentiation, matrixvesicles release, apoptosis, loss of inhibitors, and extracellularmatrix degradation (Shanahan et al., Circulation research. 2011;109:697-711). Sortilin 1 has been recognized as a crucial component ofthe signalling complex that controls survival of neurons (Jansen et al.,Nature neuroscience. 2007; 10:1449-1457 and Vaegter et al., Natureneuroscience. 2011; 14:54-61). However, in the present study modulationof sortilin 1 did not affect cell viability. Evidence demonstrates thatSortilin 1 assists in sorting of target proteins in the secretory and/orthe endosomal pathway. Sortilin 1 was shown to bind to lipoproteinlipase, and mediated endocytosis (Nielsen et al., The Journal ofbiological chemistry. 1999; 274:8832-8836); thus promoting osteogenesisand inhibiting adipogenesis of mesenchymal stem cells (Maeda et al.,Journal of cellular physiology. 2002; 193:73-79). In addition, sortilin1 has been identified to bind TGF-β family precursor proteins andpromote their trafficking to the lysosome for degradation (Kwon et al.,The Journal of biological chemistry. 2011; 286:21876-21885). TGF-β playsa crucial role in bone matrix production by a high-phosphateenvironment. Thus, it is reasonable to propose that sortilin 1 binds,sorts and degrade triggering molecules, including calcificationinhibitors, and thereby promote vascular calcification.

Without wishing to be bound by a theory, based on our evidence thatexogenous sortilin 1 increases matrix mineralization and serum sortilin1 levels are elevated in atherosclerotic mice in our study, circulatingsortilin 1 acts as a ligand for unknown cell-surface receptor or as adecoy receptor, which can trigger intracellular events leading tocalcification. Indeed, shedding of luminal domain byzinc-metalloproteases and ADAM10 resulted in a soluble form of sortilin1 (Navarro et al., Biochemical and biophysical research communications.2002; 298:760-764). Hence, neutralizing antibodies can be used tointerfere the sortilin 1-mediated pro-calcific pathway. However, whilewe found increased levels of sortilin 1 in mouse and human blood, nopublication has previously demonstrated soluble sortilin 1 levels inhuman blood.

MiRNAs have been identified to play key roles in cardiovascular diseases(Creemers et al., Circulation research. 2012; 110:483-495). Previously,we demonstrated a role of miR-125b in osteogenic transition of SMCs. Inthe current study, we found that miR-125b has a binding site at the 3′end of SORT1. Indeed, miR-125b directly binds to sortilin 1, asdemonstrated in the present study. Decreased expression of miR-125b incalcified SMCs (Goettsch et al., The American journal of pathology.2011; 179:1594-1600) increased sortilin 1. Our study thus identifiedmiR-125b as a suppressor for sortilin 1 expression.

Our current investigations into the sortilin 1 co-IP proteome aims tonot only characterize further binding partners of sortilin 1, but alsoto differentiate between activity-dependent partners and those which aredirect targets for subcellular trafficking, in the context of SMCcalcification. Our data demonstrates a role for subcellular traffickingin sortilin 1-dependent calcification events. Given that we have accessto state-of-the-art mass spectrometry strategies, we are enabled withnot only identification based methods, but also a suite of quantitativemethods to conduct in-depth biochemical studies on sortilin 1. Such MSworkflows can determine, for example, absolute levels of circulatingsortilin 1^(42,43); the relative stoichiometries of sortilin 1 bindingpartners/substrates (Singh et al., Journal of proteome research. 2009;8:2201-2210), and even quantitative mapping of potentialcalcification-dependent post-translational events on sortilin 1 (Singhet al., Flexiqinase, a mass spectrometry-based assay, to unveilmultikinase mechanisms. Nature methods. 2012; 9:504-508).

In conclusion, our findings demonstrate a novel mechanism thataccelerates cardiovascular calcification in CRD via interaction ofmiR-125b and SORT1, as illustrated in FIG. 6. Our experiments haveunravelled the role of sortilin 1 as a novel regulator ofhyperphosphatemia-triggered osteogenic vascular SMC transition andcalcification, proving its role as a therapeutic target.

In calcified regions of human atherosclerotic plaques, cellsco-expressed sortilin 1 and activated Runx2, a regulator of osteoblastdifferentiation. In human SMCs osteogenic phosphate-rich media inducedan osteoblast-like phenotype, coinciding with a 22-fold increasedexpression of sortilin 1 mRNA/protein. Silencing of sortilin 1 by siRNAsignificantly reduced alkaline phosphatase activity (TNAP) (30%) andmatrix mineralization (33%) in calcified SMCs. In contrast, increasedendogenous or exogenous sortilin 1 promoted TNAP activity by 37% andmatrix mineralization by up to 77%. PCR array revealed a significantinverse correlation between phosphate-regulating endopeptidase (PHEX)and sortilin 1. Sortilin 1 siRNA induced PHEX by 1.5-fold, whereasincreased sortilin 1 diminished PHEX expression by 77%. In silicoanalysis suggested that sortilin 1 is a target for microRNA-125b. Weverified miR-125b indeed binds to 3′UTR of SORT1, repressing itsexpression. Analysis of the sortilin 1 co-immunoprecipitated proteomeidentified candidate binding proteins that could be differentiallyregulated as a function of SMCs calcification. Induction of CRD by 5/6nephrectomy in Apoe−/− mice increased serum phosphate and sortilin 1serum levels by 2.8-fold and 3.3-fold, respectively, and showed asignificant correlation between these two factors. Moreover, CRDdramatically increased sortilin 1 expression in medial SMCs (+667%).

Vascular calcification is a prominent feature of chronic inflammatorydisorders such as chronic renal disease (CRD) and atherosclerosis, andhas no medical therapies. Human genome wide association studies linkedthe SORT1 gene, encoding sortilin 1, with increased risk ofcardiovascular diseases and coronary artery calcification; however,underlying mechanisms are unknown. The work reported herein demonstratesthat sortilin 1 contributes to the osteogenic transition of vascularsmooth muscle cells (SMC). The current study shows for the first time adirect role of sortilin 1 in vascular calcification, demonstrating thatsortilin 1 is a therapeutic target for patients with CRD.

Genome wide association studies (GWAS) have strongly associated the 1p13locus harbouring the SORT1 gene that encodes sortilin 1 with plasmalow-density lipoprotein (LDL) cholesterol levels, the onset ofmyocardial infarction and coronary artery calcification (Musunuru K etal, Nature, 2010; Kathiresan S et al, Nature Genetics, 2009; O'Donnell CJ et al, Circulation, 2011). Furthermore, the global absence of Sort1reduces secretion of lipoproteins from the liver and ameliorateshypercholesterolemia and atherosclerosis in LDL receptor-deficient mice(Kjolby M et al, Cell Metabolism, 2010). It is a novel finding by theinventors that sortilin 1 plays a direct role in vascular calcification.In calcified regions of human atherosclerotic plaques, cells co-expresssortilin 1 and activate RUNX2, a regulator of bone osteoblastdifferentiation. In human SMC osteogenic phosphate-rich media induce anosteoblast-like phenotype, coinciding with an increase expression ofsortilin 1 mRNA/protein. Silencing of sortilin 1 significantly reducecalcification of SMC measured by the alkaline phosphatase activity andamount of matrix-calcium. In contrast, increase endogenous or exogenoussortilin 1 promotes SMC calcification. Using proteomics approach weidentified candidate binding proteins that differentially regulated as afunction of SMC calcification. The association of sortilin 1 tocytosolic proteins (e.g. myosin 9) gets lost in calcified SMC, whereasthe association to alkaline phosphatase, caveolin-1, erlin-1 andcathepsin-D was strongly increased in calcified SMC. Induction of CRD by5/6 nephrectomy in Apoe-deficient mice increases serum phosphate andsortilin 1 serum levels and show a significant correlation between thesetwo factors. Moreover, CRD dramatically increases sortilin 1 expressionin medial SMC. Collectively, the work reported herein shows thatsortilin 1 is a novel regulator of hyperphosphatemia-triggeredosteogenic vascular SMC transition and calcification. This is the firstreport to inventors' knowledge to demonstrate a direct role of sortilin1 in vascular calcification.

Our study demonstrates a novel finding that sortilin 1 is present incalcified atherosclerotic plaques expressed by osteogenic cells withinthe lesion. Inhibition of sortilin 1 prevents mineralization of SMC invitro. In vivo, in a mouse model of CRD sortilin 1 was highly increasedin calcified vessel media and intima. Serum sortilin 1 levels wereincreased in CRD mice and correlate positively with phosphate levels.

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

1. A method for inhibiting calcification of a smooth muscle cell (SMC),the method comprising contacting a compound with a SMC, wherein thecompound inhibits: (i) activity or amount of sortilin 1 in a smoothmuscle cell; or (ii) expression of a nucleic acid encoding sortilin 1 ina smooth muscle cell.
 2. The method of claim 1, the compound is selectedfrom the group consisting of small organic or inorganic molecules;saccharines; oligosaccharides; polysaccharides; peptides; proteins;peptide analogs and derivatives; peptidomimetics; glycoproteins,glycopeptides; antibodies and antigen binding fragments thereof; nucleicacids; nucleic acid analogs and derivatives; an extract made frombiological materials such as bacteria, plants, fungi, or animal cells;animal tissues; naturally occurring or synthetic compositions; and anycombinations thereof.
 3. The method of claim 2, wherein the compound isa siRNA or an antibody.
 4. The method of claim 1, wherein the compound:(i) inhibits the expression of the nucleic acid encoding sortilin 1 byat least 10% relative to a control or reference level; (ii) inhibits theactivity of sortilin 1 by at least 10% relative to a control orreference level; (iii) decreases the amount of sortilin 1 in the SMC byat least 10% relative to a control or reference level; (iv) reducestissue non-specific alkaline phosphatase activity (TNAP) by at least 10%relative to a control or reference level; (v) increases phosphateregulating endopeptidase (PHEX) expression by at least 10% relative to acontrol or reference level; (vi) decreases matrix mineralization by atleast 10% relative to a control or reference level; (vii) increasesexpression level of microRNA 125b by at least 10% relative to a controlor reference level; or (viii) decreases the association of sortilin 1 tonon-specific alkaline phosphatase activity (TNAP) or Caveolin-1.
 6. Themethod of claim 1, wherein said contacting is in a subject in need ofinhibition of calcification.
 7. The method of claim 6, wherein saidcalcification is cardiovascular calcification.
 8. The method of claim 6,wherein said calcification is valvular or arterial calcification.
 9. Anassay for determining calcification in a subject, the assay comprisingsubjecting a biological sample of a subject to at least one analysis todetermine level of sortilin 1 in the biological sample, wherein anincreased level of sortilin 1 relative to a reference level indicatesthe subject has calcification, is at risk of developing calcification,has CRD, or is at risk of developing CRD, and wherein said at least oneanalysis is selected from the group consisting of western blot, enzymelinked absorbance assay, mass spectrometry, immunoassay, flow cytometry,immunohistochemical analysis, probe hybridization, primer extension,amplification, sequencing, 5′ nuclease digestion, molecular beaconassay, DNA chip analysis, oligonucleotide ligation assay, size analysis,single-stranded conformation polymorphism, polymerase chain reaction(PCR), real-time quantitative PCR, and any combinations thereof.